(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Novel fucosylation pathway in the cytosol of Dictyostelium discoideum"

A NOVEL FUCOSYLATION PATHWAY IN THE CYTOSOL 
OF DICTYOSTELIUM DISCOIDEUM 



By 
BEATRIZ GONZALEZ -YANES 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 
OF THE REQUIREMENTS FOR THE DEGREE OF 
DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1991 






A mis padres 
(To my parents) 



ACKNOWLEDGEMENTS 

First of all I would like to acknowledge my advisor. 
Dr. Christopher M. West, for his time, training, and example 
throughout these studies. I would also like to thank the 
members of my committee, Drs . Robert Cohen, William Dunn, 
and Carl Feldherr, for their time and guidance regarding 
this project. 

I would like to acknowledge and thank Dr. Michael Ross 
for his interest and support of my graduate education. 

Part of the work presented in Chapter II is reproduced 
from the journal of Developmental Biology, 1989, volume 133, 
pages 576-587 by copyright permission of Academic Press, 
Inc. 

I am very grateful to the following researchers and the 
members of their laboratories for sharing equipment and/or 
reagents with us: Drs. Gudrun Bennett, Ross Brown and Lina 
Gritzali, William Dunn, Carl Feldherr, Michael Humphreys- 
Beher, K.J. Kao, and Gillian Small. 

In general, I would like to express my gratitude to the 
faculty and staff in the Department of Anatomy and Cell 
Biology for their support and interest in my advancement as 
a scientific investigator. I would like to acknowledge Kari 
Eissinger for her help in photographing and printing some of 

iii 



the figures contained herein. Particularly, I would like to 
thank Drs . Kelly Selman and Gillian Small for the advice and 
friendship they have provided during these years. I am also 
grateful to have been in contact with other students, past 
and present, which have made the time spent in the 
department more enjoyable. I am grateful to Dan Tuttle for 
reviewing my dissertation. I would also like to acknowledge 
all the people that have worked in the laboratory of Dr. 
West, including Scherwin Henry, for making the time spent in 
the laboratory more rewarding and Mel Fields for his 
assistance in some experiments. 

I would like to acknowledge my professors at the 
University of Puerto Rico, who encouraged me to pursue a 
career in biological research. 

I appreciate all the friends I have made in Gainesville 
in the past years and would like to acknowledge their 
importance in my education. Especially, I thank Hans van 
Oostrom for his moral support, encouragement, and for 
teaching me about the wonders of the electronic world and 
those rare creatures called computers. 

Finally, and most importantly, I would like to thank my 
family. I thank my grandparents, especially Abuela Emma and 
Abuela Yuya, for the faith they have showed in me and for 
their prayers, which have helped me through my graduate 
education. My brothers German, Omar, and Carlos have been 
great sources of happiness and pride. Lastly, I thank my 

iv 



parents, Beatriz and German, for everything. I firmly 
believe that the education received during my first twenty- 
one years of life in their company is ultimately responsible 
for every achievement in my life. 



TABLE OF CONTENTS 

ACKNOWLEDGEMENTS iii 

LIST OF TABLES viii 

LIST OF FIGURES ix 

LIST OF ABBREVIATIONS xi 

ABSTRACT xiv 

CHAPTERS 

I HISTORICAL REVIEW AND BACKGROUND 1 

Introduction 1 

Fucosylated Macromolecules 3 

Fucose-Binding Proteins 13 

II CHARACTERIZATION OF A FUCOSYLATION MUTANT .... 18 

Introduction 18 

Materials and Methods 19 

Results 24 

Discussion 40 

III IDENTIFICATION OF A CYTOSOLIC FUCOPROTEIN .... 45 

Introduction 45 

Materials and Methods 46 

Results 54 

Discussion 83 

IV EVIDENCE FOR A CYTOSOLIC FUCOSYLTRANSFERASE ... 90 

Introduction 90 

Materials and Methods 92 

Results 98 

Discussion 135 

V SUMMARY AND CONCLUSIONS 144 

Summary of Results 144 

Future Studies 149 

vi 



REFERENCES 156 

BIOGRAPHICAL SKETCH 166 



vii 



LIST OF TABLES 

Table Page 

2-1. Fucose content of normal and mutant spores and 

vegetative cells 29 

2-2. Effect of time on conversion of GDP-mannose to 

GDP-fucose 34 

2-3. Specific activities of fucose 39 

3-1. Distribution of protein and radioactivity in S100 

and P100 fractions 56 

3-2. Radioactivity recovered in the second S100 after 

different P100 treatments 65 

3-3. Distribution of markers among S100 and P100 

fractions 66 

4-1. Effect of different treatments on the cytosolic 

fucosyltransferase activity 102 

4-2. Failure to sediment S100 fucosyltransferase 

activity 104 

4-3. Comparison between the S100 and P100 

fucosyltransferase activities 112 

4-4. Fucosyltransferase activity in Ax3 S100 fraction . 122 

4-5. Utilization of 8-methoxycarbonyloctyl synthetic 

acceptors by cytosolic fucosyltransferase activity 
from Ax3 and HL250 128 

4-6. Evaluation of the suitability of p-nitro-phenyl 
glycosides as acceptors for cytosolic fucosyl- 
transferase activities in HL250 and Ax3 S100 . . . 132 

4-7. Reduction of fucosylation of acceptor type I analog 

by purified FP21 134 

4-8. In vitro fucosyltransferase activity of HL250 and 

Ax3 slug extracts 136 



vm 



LIST OF FIGURES 

Figure Page 

2-1. Localization of SP96 in prespore and spore cells . 27 

2-2. Biosynthesis of GDP-L-fucose 32 

2-3. Conversion of GDP-mannose to GDP-fucose by normal 

and mutant strains 37 

3-1. Incorporation of [ 3 H]fucose into macromolecular 

species of the S100 and P100 58 

3-2. Proteinaceous nature of FP21 60 

3-3. Comparison of S100 and releasable P100 components . 63 

3-4. Gel filtration chromatography of FP21 

glycopeptides 71 

3-5. Gel filtration chromatography of PNGase F digests . 74 

3-6. Gel filtration chromatography of FP21 

oligosaccharides 77 

3-7. Gel filtration chromatography of P100 

glycopeptides 81 

3-8. Incorporation of [ HJfucose into macromolecular 

species of slug stage cells 85 

4-1. Fucosylation of endogenous acceptors by S100 

fraction 101 

4-2. SDS-PAGE profile of endogenous acceptors 

fucosylated in vitro 107 

4-3. BioGel P-4 gel filtration chromatography of in 

vitro labelled FP21 oligosaccharide 110 

4-4. Effect of pH on S100 and P100 fucosyltransf erase 

activities in the presence of Tween-20 115 



ix 



4-5. Effect of GDP-fucose concentration on S100 and P100 
fucosyltransferase activities in the presence of 
Tween-20 117 

4-6. Effect of GDP-fucose concentration on intact S100 

and P100 fucosyltransferase activities 120 

4-7. Fucosylation of mutant FP21 by Ax3 S100 fraction . 126 

4-8. Haworth projections of the structures of the synthetic 
glycolipid acceptors 130 



APA 
ATP 
BSA 

14 c 

CDNA 

cm 

Con A 

cpm 

CHO 

dH 2 

dpm 

EDTA 

EtOH 

GlcNAc 

GDP 

h 

3 H 

HMG 

HPLC 

kD 

K 

m 
1 



LIST OF ABBREVIATIONS 
Asparagus Pea Agglutinin 
Adenosine-5 ' -triphosphate 
Bovine Serum Albumin 
Radioactive Carbon 

Complementary Deoxyribonucleic Acid 
Centimeter (s) 
Concanavalin Agglutinin 
Counts per Minute 
Chinese Hamster Ovary 
Distilled Water 
Disintegrations per Minute 
Ethylenediaminetetraacetic Acid 
Ethanol 

N-acetyl Glucosamine 
Guanosine-5 ' -diphosphate 
Hour(s) 
Tritiated 

High Mobility Group 

High Performance Liquid Chromatography 
Kilodaltons 
Michaelis Constant 
Liter(s) 



xi 



M 

mCi 

mAb 

MES 

mg 

ml 

min 

mm 

mM 

MW 

juCi 

jl/M 

nm 

P 

PAGE 

pmol 

PMSF 

PNGase F 

Rev 

RNAse B 

SDS 

TCA 

Tris 

U 

UEA-I 

Ve 



Molar Concentration 

Millicurie(s) 

Monoclonal Antibody 

2-(N-Morpholino)ethanesulfonic Acid 

Milligram(s) 

Milliliter(s) 

Minute(s) 

Millimeter(s) 

Millimolar 

Molecular Weight 

Microcurie(s) 

Micromolar 

Nanometers 

probability 

Polyacrylamide Gel Electrophoresis 

Picomole(s) 

Phenylmethylsulfonyl Fluoride 

Peptide N-glycosidase F 

Relative Elution Coefficient 

Ribonuclease B 

Sodium Dodecyl Sulphate 

Trichloroacetic Acid 

Tris ( hydroxymethyl ) aminomethane 

Unit(s) 

Ulex europaeus Agglutinin 

Elution Volume 



XII 



Vi Inclusion Volume 

V Maximal Velocity 

max 

Vo Void volume 

v/v Volume per Volume 

WGA Wheat Germ Agglutinin 

w/v Weight per Volume 



Xlll 






Abstract of Dissertation Presented to the Graduate School 
of the University Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 

A NOVEL FUCOSYLATION PATHWAY IN THE CYTOSOL 
OF DICTYOSTELIUM DISCOIDEUM 

By 

Beatriz Gonzalez-Yanes 

December 1991 

Chairman: Dr. Christopher M. West 

Major Department: Anatomy and Cell Biology 

The existence of a fucosylation pathway in the cytosol 

of Dictyostelium discoideum was investigated in the 

glycosylation mutant strain, HL250, and the normal parental 

strain, Ax3. HL250 was characterized as a conditional 

mutant that cannot convert GDP ( guanosine-5 ' -diphosphate) - 

mannose to GDP-fucose, resulting in a lack of macromolecular 

fucosylation unless grown in the presence of extracellular 

fucose. HL250 or Ax3 cells were metabolically labelled with 

[ 3 H]fucose, filter-lysed, and fractionated by high speed 

centrifugation into sedimentable (P100) and soluble (S100) 

fractions. The fractions exhibited unique profiles of 

fucoconjugates as analyzed by gel electrophoresis. The 

major acceptor in the S100 was a 21 kilodalton molecular 

weight protein, FP21. Analysis of FP21 oligosaccharide by 

mild alkaline hydrolysis and gel filtration chromatography 

xiv 



revealed that fucose was incorporated into an O-linked 
oligosaccharide with an average size of 4.8 glucose units. 
FP21 appeared to be endogenous to the cytosol, based on the 
failure to release FP21 from P100 vesicles by sonication, 
and the absence of FP21-like glycopeptides derived by 
pronase digestion of the P100. 

To determine if FP21 was fucosylated in the cytosol, 
S100 and P100 fractions from HL250 were assayed for 
fucosyltransferase activity, measured as ability to transfer 
[ 14 C] from GDP- [ 14 C] fucose to endogenous acceptors. 
Fucosylation of FP21 by the S100 was time- and protein 
concentration-dependent. The cytosolic activity was 
distinguished from the bulk P100 activity by its absolute 
divalent cation dependence, alkaline pH sensitivity, and 
very low apparent K for GDP- fucose. Fucosyltransferase 
activity was not detectable in Ax3 cytosol. However, 
activity was reconstituted by addition of purified mutant 
FP21, suggesting FP21 was already fucosylated in living 
cells, and Ax3 possessed a cytosolic fucosyltransferase 
equivalent to the HL250 enzyme. S100 fractions from Ax3 and 
HL250 were able to fucosylate an al,4fucosyltransferase- 
specific acceptor glycolipid analog, but not analogs capable 
of being modified by otl,2 and crl,3 f ucosyltransf erases . 
Since fucosylation of the analog was reduced by addition of 
purified FP21, the same enzyme appears to be responsible for 
fucosylation of both molecules. Thus it is proposed that 

xv 



FP21 is synthesized and fucosylated in the cytosol by an 
al , 4 f ucosyltransf erase . 



xvi 



CHAPTER I 
HISTORICAL REVIEW AND BACKGROUND 



Introduction 
Glycoprotein synthesis and localization have been 
subjects of intense study in the last few decades. Much has 
been learned about glycosylation and some excellent reviews 
are available (Kornfeld and Kornfeld, 1985; Hirschberg and 
Snider, 1987). Carbohydrate moieties are usually 
categorized as N and/or O-linked depending on whether the 
carbohydrate moiety is attached to an asparagine by an amide 
glycosidic linkage or to a serine or threonine, 
respectively. Glycosylation is generally considered to be a 
modification restricted to macromolecules that pass through 
the secretory pathway, starting in the rough endoplasmic 
reticulum where N-linked glycosylation is initiated 
(Kornfeld and Kornfeld, 1985), but in some instances 0- 
linked glycosylation also takes occurs (Spielman et al., 
1988). Glycosylation then continues in the Golgi apparatus 
where further processing of N-linked oligosaccharides may 
occur and O-linked glycosylation takes place (Abeijon and 
Hirschberg, 1987). In the case of fucosylation, L-fucose 
has been shown to be added as a terminal modification to 
either N-linked or O-linked oligosaccharides in the Golgi 



2 

apparatus utilizing GDP-fucose as the sugar nucleotide donor 
(Bennett et al., 1974; Hirschberg and Snider, 1987). 
However, there are a few exceptions to these remarks, since 
fucose has been found to be attached directly to serine and 
threonine, although the site for this modification has not 
been identified (Hallgreen et al., 1975), and to be present 
in homopolymers , as in fucoidans (Flowers, 1981). It has 
been suggested that fucosylation also occurs in the 
endoplasmic reticulum as well as in the Golgi apparatus in 
thyrotrophs under different physiological conditions (Magner 
et al., 1986). There are two pathways for the biosynthesis 
of GDP-fucose. The main source of GDP-fucose is the 
conversion pathway of GDP-mannose to GDP-fucose (Yurchenco 
et al., 1978; Flowers, 1985). Alternatively, synthesis of 
GDP-fucose may occur by the fucose salvage pathway in the 
presence of extracellular L-fucose (Yurchenco et al., 1978; 
Flowers, 1985; Ripka and Stanley, 1986; Reitman et al., 
1980) . 

Glycosylation is carried out by diverse specific 
glycosyl transferases which have been located in the 
endoplasmic reticulum and Golgi apparatus. One underlying 
assumption is that the acceptor macromolecule must 
colocalize with the transferase enzyme in order to be 
glycosylated. However, there have been occasional reports 
of glycoproteins in compartments topologically discontinuous 
with the lumen of the rough endoplasmic reticulum and Golgi 



3 
apparatus. These findings contradict the dogma that 
glycosylation is strictly an endoplasmic reticulum- and 
Golgi apparatus -dependent event. Two models could account 
for the existence of glycoproteins outside the realms of the 
secretory pathway; one postulates that lumenal glycosylated 
proteins translocate across the membrane back to the 
cytosolic space, the other that glycosyltransferases are 
localized outside of the lumen of the endoplasmic reticulum 
or Golgi apparatus. Evidence has been accumulating in the 
past decade that supports the latter model. In this chapter 
I shall be concerned with the presence of fucosylated 
proteins in compartments topologically discontinuous with 
the lumen of the endoplasmic reticulum and Golgi apparatus. 
Subseguently, I shall analyze some of the studies that 
suggest the presence of fucosyltransferases that would co- 
compartmentalize with such fucoproteins . In light of the 
results presented in this dissertation, a review focussing 
on the presence of fucoproteins and fucosyltransferases 
outside the secretory pathway will be useful. An excellent 
review is available that examines nuclear and cytosolic 
glycosylation in general (Hart et al., 1989a). 

Fucosylated Macromolecules 
The presence of fucosylated proteins in compartments 
discontinuous with the secretory pathway has been documented 
since the 1970' s. Various technigues have been employed in 



4 
these reports, including the direct analysis of carbohydrate 
content, use of lectins, use of radiolabeled fucose and/or 
a combination of biochemical and morphological techniques. 

Nuclear Fucosylated Macromolecules 

In contrast to the prevailing dogma, fucosylated 
macromolecules have been detected in the nucleus. Such 
studies demonstrated lectin binding to nuclear membranes, 
chromatin, nuclear proteins, and the nuclear matrix. These 
studies generally involved localization by binding of 
labelled lectins (fluorescent, radiolabeled, ferritin- 
conjugated, etc.) to isolated nuclei. Binding specificity 
was assessed by competition of labelling with hapten 
inhibitors. Occasionally, these studies were supplemented 
by metabolic labelling experiments with radioactive fucose 
which is primarily incorporated into fucoproteins with 
little metabolism into other molecular species in eukaryotic 
cells (Yurchenko et al., 1978). 

Nuclear membranes . One early report suggested the 
presence of fucose-containing structures on the cytoplasmic 
face of isolated bovine nuclei (Nicolson et al., 1972). 
Nicolson et al. (1972) found that purified nuclei were 
agglutinated by the L-fucose-specific lectin UEA-I from Ulex 
europaeus (Lis and Sharon, 1986), and agglutination was 
inhibited by incubation in the presence of L-fucose, 



5 
suggesting that there were fucose-containing membrane-bound 
oligosaccharides on the outer nuclear membrane. Similar 
results were obtained with the mannose- and glucose-specific 
lectin concanavalin A (Con A), which was also found to 
agglutinate purified nuclei (Nicolson et al., 1972). 
However, subseguent work by another group revealed that, as 
evidenced by electron microscopic examination, ferritin-Con 
A appeared to stain only damaged nuclei (Virtanen and 
Wartiovaara, 1976) raising some concerns about the studies 
by Nicolson et al. (1972). Likewise, in the aforementioned 
studies (Nicolson et al., 1972), integrity of the isolated 
nuclei was not determined, allowing for the possibility that 
the lectin was interacting with lumenal fucoproteins that 
escaped organelles during nuclei isolation or with 
contaminating fucoprotein-containing membranes, such as 
plasma membrane. 

Chromatin . The existence of chromatin associated 
fucose-containing proteins has been suggested by several 
laboratories. Early on, Stein et al. (1975) reported the 
presence of [ 3 H] labelled glycoconjugates in purified 
chromatin from HeLa cells grown in the presence of 
[ 3 H]fucose. Although the label was not examined to 
corroborate its presence as fucose, in the case of 
radiolabelling macromolecules with radioactive sugar 
precursors, fucose is an excellent candidate because it has 






6 
been shown to be incorporated as such, with minimal 
metabolizing of the label (Yurchenko et al., 1978). In 
these studies the authors examined a very pure chromatin 
preparation, with essentially no contamination from nuclear 
or plasma membranes. The authors argued against plasma 
membrane contamination based on mixing experiments, in which 
radiolabeled cell-surface trypsinates were combined with 
unlabeled nuclear preparations prior to chromatin isolation. 
The fucosylated chromatin-associated macromolecules were 
deemed to be fucoproteins based on their sensitivity to 
pronase digestion. Unfortunately, these studies have not 
been pursued further, and many questions remain unanswered 
with respect to their structure and biosynthesis. 

Chromatin-associated fucoproteins were also detected in 
normal rat liver and Novikoff hepatoma ascites cells 
(Goldberg et al., 1978) using the L-fucose-specif ic lectin 
asparagus pea agglutinin (APA) . The authors reported a 
strongly basic fucoprotein, that was sensitive to pronase 
digestion. Based on the reactivity with the lectin, they 
calculated that the protein was three times more 
concentrated in tumor chromatin than in normal liver cells. 
However, the method for chromatin purification did not 
eliminate adequately nuclear inner membrane contamination, 
and since binding to the lectin was assayed by 
af finoelectrophoresis a molecular weight for the protein was 
not determined (Goldberg et al., 1978). 



7 
In a more recent study on duodenal columnar cells, Kan 
and Pinto da Silva (1986) used UEA-I conjugated to colloidal 
gold in freeze- fracture electron microscopy of cross- 
fractured nuclei. They compared binding of the conjugated 
lectin to euchromatin, heterochromatin, and nucleolus; 
compartments which can be differentiated ultrastructurally. 
Binding of UEA-I showed that colloidal gold particles were 
almost exclusively confined to cross-fractured areas where 
euchromatin was exposed. Labelling was abolished by 
pretreatment and incubation in the presence of L-fucose, as 
expected for a specific label. Pre-digestion of the 
fractions with trypsin also abolished labelling, suggesting 
the receptors for UEA-I binding were glycoproteins. The 
preferential binding to euchromatin may be of importance, 
because replication and transcription take place at 
euchromatin regions. Although the results reported are 
intriguing, the authors did not identify the type of fucose- 
containing proteins detected. DNA-associated proteins can 
be classified as either histone or non-histone proteins, and 
as summarized below, both types of proteins appear to be 
fucosylated. 

Histories. The his tones are the most abundant proteins 
associated with DNA. Histones are very basic proteins and 
are found in all nuclei (Darnell et al., 1986). Based on 
the specific binding to UEA-I, Levy-Wilson (1983) presented 



8 

evidence that histones isolated from the macronucleus of 
Tetrahymena thermophila appear to contain fucose. These 
results were strengthened by metabolic incorporation of 
[ 3 H] fucose into histones, which showed that all five 
Tetrahymena histones, HI, H2A, H2B, H3, and H4, appear to 
contain fucose, with H2A incorporating the highest amount 
(Levy-Wilson, 1983). In these studies macronuclei were 
isolated to high purity, and highly pure histone 
preparations were obtained after extensive washing in high 
salt to remove nonhistone proteins. Using Con A, the author 
showed specific binding to histones, which was interpreted 
as evidence for the presence of mannose residues. However, 
Con A has previously been shown to also recognize D-glucose 
residues. Based on the extent of fucose incorporation and 
its specific radioactivity, the author estimated, as the 
lowest estimate, that one in a thousand nucleosomes 
contained a fucosylated H2A molecule. To date, the 
glycosylation pathway of histones and the oligosaccharide 
structure(s) present in histones remain unknown. 

Nonhistone proteins . High mobility group (HMG) 
proteins are fairly abundant nonhistone chromosomal proteins 
classified according to their relative electrophoretic 
mobilities (Darnell et al . , 1986). HMG proteins undergo a 
variety of posttranslational modifications, including 
glycosylation. Since they appear to be preferentially 



9 
associated with actively transcribed DNA, it has been 
speculated that glycosylation may influence gene activity. 
Highly purified HMG14 and HMG17 from mouse Friend 
erythroleukemic cells were found to contain fucose, among 
other sugars, by direct composition analysis (Reeves et al., 
1981). These proteins bound UEA-I specifically and could be 
metabolically labelled with [ 3 H] fucose. The 
oligosaccharides were largely insensitive to fi-elimination, 
suggesting an N-linkage to protein (Reeves et al . , 1981). 
When purified HMG14 and HMG17 were digested with mixed 
glycosidases, the binding of HMG14 and HMG17 to the nuclear 
matrix was abolished. Even though they did not employ a 
fucosidase, making it difficult to ascertain the role of 
fucose in binding, it was evident that glycosylation 
influenced binding to the nuclear matrix (Reeves and Chang, 
1983). These studies presented convincing evidence that HMG 
14 and 17 are fucosylated, although they did not explore the 
site of modification or the composition of the 
oligosaccharide^ ) . 

Nuclear fucoproteins and transcriptional activity . 
Since some glycoproteins have been found associated with DNA 
there has been speculation about a possible correlation 
between the state of nuclear glycosylation and 
transcriptional activity (Hart et al., 1989a). There are 
examples in the literature of glycosylated transcription 



10 
factors and, at least in one case, glycosylation may have 
influenced transcriptional activity (Lichtsteiner and 
Schibler, 1989; Jackson and Tjian, 1989). As mentioned 
earlier, in the case of fucosylated macromolecules, it was 
found that in Novikoff hepatoma cells chromatin had three 
times the amount of a fucoprotein of normal liver cells 
based on APA binding (Goldberg et al., 1978). However, the 
identity, size, or number of such proteins were not well 
documented. Putative fucoproteins were found preferentially 
associated with euchromatin (Kan and Pinto da Silva, 1986). 
Fucosylated histones were found in the macronucleus of 
Tetrahymena , where transcriptionally active chromatin is 
compartmentalized (Levy-Wilson, 1983). Nevertheless, 
histones from non-active chromatin were not studied, so no 
comparisons can be made with heterochromatin. Levy-Wilson 
(1983) suggested that the reason why other investigators 
have failed to detect fucosylation in mammalian histones is 
due to the low proportion of transcriptionally active genome 
which would imply a low concentration of fucosylated 
histones. 

Although the data gathered in these reports are 
interesting, they are the result of isolated studies from 
diverse organisms and it is difficult to draw generalized 
conclusions. In addition, virtually nothing is known about 
the structure or biosynthesis of the fucose-containing 
moieties. Even though the fractions in all these reports 



11 

appeared to have almost no contamination, structural studies 
showing a different glycocon jugate from those found in other 
organelles would convincingly argue against contamination. 
Nevertheless, these provocative studies are encouraging and 
deserve to be pursued further. 

Cytosolic Fucoconjugate 

The existence of glycoproteins in the cytosol has been 
documented in the past and reviewed recently (Hart et al., 
1989a). Studies suggesting the existence of glycoproteins 
in the cytosol were based on determinations of lectin 
binding sites or biochemical compositional analyses (Hart et 
al., 1989a). Some of the negative results reported by those 
studies, that relied on lectin binding as confirmation for 
the presence of a sugar residue, may be misleading since 
lack of binding may reflect a poor choice of lectins to 
probe with and not necessarily the absence of a fucose 
residue. While there are several fucose-binding lectins 
available that serve as useful biochemical tools, they do 
not recognize every possible fucose-containing structure. 
Lectin binding is dependent on a specific array of sugar 
residues, and does not depend solely on the presence of 
fucose (Lis and Sharon, 1986). However, as will be 
described below, there is one biochemical study that reports 
the existence of a cytosolic fucoconjugate. In many cell 
fractionation experiments, the cytosolic compartment is 



12 
defined by the lack of sedimentation during high-speed 
centrifugation. However, this criterion alone is not 
sufficient, since cytosolic glycans might arise from 
contamination by other organelles. The most convincing 
evidence describes fucoconjugates that appear to be 
preferentially enriched in the cytosol in relation to other 
compartments . 

In studies in rat brain, a soluble proteoglycan that 
contains novel O-linked mannose-containing oligosaccharides 
was recovered in the cytosolic fraction (Margolis et al., 
1976; Finne et al., 1979). The proteoglycan was 
characterized as a soluble chondroitin sulfate proteoglycan, 
that contained neutral oligosaccharides releasable by mild 
alkaline borohydride treatment. The oligosaccharides 
contained mannose at their proximal ends, and one 
oligosaccharide was proposed to be composed of mannose, 
GlcNAc, fucose, and galactose (Finne et al., 1979). 
However, the possibility remains that the oligosaccharides 
are not integral components of the proteoglycan, but were 
associated with other co-purified material. Nevertheless, 
the oligosaccharides appeared to be endogenous to the 
cytosol and not the result of contamination from other 
fractions, since they were present in only trace amounts in 
the microsomal or synaptosomal membrane fractions (Finne et 
al . , 1979). Unfortunately, the function and biosynthetic 
pathway of these oligosaccharides remain unknown. 



13 
Fucose-Bindinq Proteins 
The presence of fucoproteins in the nucleus and cytosol 
prompted the idea that there might be specific proteins 
inside the cell that bind, and/or modify these fucoproteins, 
as is the case with the fucoproteins in the secretory 
pathway. This would include fucose-binding proteins, 
fucosyltransferases responsible for fucosylation, and 
fucosidases responsible for fucose removal. There are some 
studies suggesting the existence of fucose-binding lectins 
and fucosyltransferases. However, there is no evidence for 
a nuclear or cytosolic fucosidase. 

Endogenous Lectins 

In light of evidence for glycoproteins in the cytosolic 
and nuclear compartments, investigators sought the existence 
of carbohydrate-binding proteins that would colocalize with 
such glycoproteins . Endogenous lectins have been detected 
in preparations of nucleoplasmic and/or cytosolic fractions 
of a variety of cells (Hart et al., 1989a). 

Aided by f luorescein-labeled neoglycoproteins, Seve et 
al. (1986) have postulated the presence of endogenous 
fucose-specific lectins. Baby hamster kidney cell nuclei 
were isolated by two different procedures, cell lysis and 
enucleation in Ficoll, to argue against contamination by 
cytoplasmic or membrane-derived components. Using 
fluorescein-labelled BSA conjugated to fucose in the order 



14 
of 20+5 sugar units per molecule, fluorescence microscopy 
experiments suggested that the majority of the binding 
appeared to be associated with nucleoli and nucleoplasmic 
ribonucleoprotein elements (Seve et al., 1986). 
Interestingly, it was shown by guantitative flow 
microfluorometry that nuclei from exponentially growing 
cells bound one order of magnitude more fucose-BSA than 
nuclei from contact-inhibited cells. In spite of these 
results, the authors acknowledge that it is impossible based 
on the data to ascribe biological roles to the nuclear 
fucose-binding sites or to conclude that they influence 
cellular physiology (Seve et al., 1986). 

In Dictyostelium discoideum a family of lectins, the 
discoidins, has been identified, and discoidin I has been 
the most extensively studied isoform. In erythrocyte 
agglutination assays, agglutination by discoidin may be 
inhibited by galactose, modified galactose residues, L- 
fucose, D-fucose, and other sugars, suggesting the existence 
of cell-surface sugar-dependent epitopes on erythrocytes 
recognized by Discoidin (Barondes and Haywood, 1979). 
Although discoidin I was originally thought to be a cell 
surface protein involved in cell-cell adhesion, it has since 
been established that it is primarily present in the cytosol 
(Erdos and Whitaker, 1983). Although galactose and modified 
galactose residues are the ligands bound by discoidin I with 
highest affinity, it is possible that a fucose-containing 



15 
macromolecule may serve as a ligand for it or that another 
lectin, possibly from the discoidin family, will be present 
in the cytosol with fucose-binding capability. 

Fucosyl transferases 

Intracellular fucosyltransferases identified to date, 
are localized in the lumen of the Golgi or possibly, 
endoplasmic reticulum. Thus the presence of fucoproteins in 
the nucleus and cytosol ic compartments poses questions 
pertaining to the mode of synthesis and/or intracellular 
transport of such glycoproteins. Early on, Kawasaki and 
Yamashina (1972) theorized, based on metabolic labelling, 
that nuclear membrane glycoproteins were not synthesized in 
and transported from microsomes, but were made in the 
nuclear membrane or its vicinity. A few reports have 
suggested the presence of glycosyltransferases in the 
nucleus, nuclear membranes, or cytosol that may be involved 
in the modification of several glycoproteins; however, none 
of these glycoproteins was reported to contain fucose 
(Richard et al., 1975; Galland et al., 1988, Haltiwanger et 
al., 1990). 

Though there is evidence for nuclear and cytosolic 
fucosylated macromolecules, to my knowledge there are no 
reports of nuclear or cytosolic fucosyltransferases. 
Louisot and collaborators reported the purification and 
separation of two soluble fucosyltransferase activities from 



16 
rat small intestinal mucosa (Martin et al., 1987). They 
report the isolation of ctl,2 and al,3/l,4fucosyltransferases 
that could be candidates for cytosolic fucosyltransferases 
based on their inability to sediment after homogenization of 
cells in 0.25 M sucrose, followed by 90 min, 200k x g 
centrifugation (Martin et al., 1987). They compare the 
al,2fucosyltransferase with the or2,6sialyltransf erase, which 
is normally a Golgi enzyme converted to a soluble form by 
cleavage of the amino terminal signal anchor to allow for 
secretion (Weinstein et al., 1987). In the case of the 
fucosyltransferases, the authors did not report using 
protease inhibitors during isolation, nor did they document 
the partitioning of cellular markers in the different 
fractions (Martin et al., 1987). The reasons the enzymes 
localize to the high speed supernatant, which would usually 
be considered as the cytosolic fraction, may include the 
breakage of the microsomal vesicles or the fact that they 
were initially present extracellularly in the body fluids of 
the mucosa. In another report these investigators tested 
for the presence of glycosyl transferases in the nucleus. In 
highly purified rat liver nuclei there was a total absence 
of fucosyl transferase activity when endogenous 
macromolecules or asialofetuin were used as acceptors 
(Richard et al., 1975). 

One explanation for the lack of evidence of nuclear 
and/or cytosolic fucosyltransferases activity in any 



17 
organism may be that there indeed are no 
fucosyltransf erases. Alternatively, if there are no 
cytosolic fucosyltransf erases, the presence of 
fucoconjugates in the cytosol or nucleus may be explained by 
membrane-associated enzymes that face the cytosolic 
compartment, as has been reported for other 
glycosyltransferases (Haltiwanger et al., 1990). In 
addition, the lack of adeguate acceptors, unfavorable 
conditions for in vitro activity, and the scarcity of 
sustained interest in the field, could account for the lack 
of evidence for such fucosyltransferase(s) that, just as is 
the case with other glycosyltransferases, are not of lumenal 
origin. 

Evidence for fucosylation, and glycosylation in 
general, has been accumulating in the past decades. Until 
recently, only sporadic reports about fucoproteins appeared 
in the literature. With the identification of newly 
reported glycoconjugates (Hart et al, 1989b), interest has 
been revived in this area of research and currently there 
appears to be much interest in the field. However, a 
careful examination and sustained interest will be necessary 
in order to elucidate the structures, and modes of 
biosynthesis and functions of nuclear and cytosolic 
fucoconjugates . 



CHAPTER II 
CHARACTERIZATION OF A FUCOSYLATION MUTANT 



Introduction 
Since fucosylation comprises numerous steps which are 
potential sites for mutations, many fucosylation mutants 
have been obtained. Fucosylation consists of the synthesis 
of the sugar nucleotide donor GDP-fucose and the transfer of 
fucose from the GDP-fucose to an acceptor (Kornfeld and 
Kornfeld, 1985). The main source of GDP-fucose is the 
conversion pathway of GDP-mannose to GDP-fucose (Yurchenco 
et al., 1978; Flowers, 1985). Alternatively, synthesis of 
GDP-fucose by the fucose salvage pathway can occur in the 
presence of extracellular L-fucose, allowing cells defective 
in the conversion pathway to phenotypically revert (Ripka 
and Stanley, 1986; Reitman et al., 1980). Lesions may 
affect the formation of GDP-fucose, the transport of GDP- 
fucose to the fucosylation compartment, the transferases 
responsible for fucosylation and/or the biosynthesis or 
transport of the acceptor species to the fucosylation 
compartment. Two mutants deficient in protein-associated 
fucose were shown to be defective in the formation of GDP- 
fucose (Reitman et al., 1980; Ripka et al., 1986). A 
Chinese hamster ovary (CHO) cell line that showed a marked 

18 



19 
reduction of incorporation of fucose into macromolecules was 
unable to synthesize complex-type N-linked oligosaccharides, 
resulting in a deficiency of acceptors (Hirschberg et al., 
1982). In another case, two glycosylation mutants were 
shown to express a fucosyltransferase activity absent in the 
parental cell line with the concomitant expression of a 
novel linkage (Campbell and Stanley, 1984). 

There are several putative glycosylation mutants. One 
of these mutants, HL250, was selected after mutagenesis of 
the parental normal strain Ax3 for the inability to bind 
anti-SP96 antiserum (Loomis, 1987). This mutant also failed 
to express a fucose-dependent epitope recognized by the 
monoclonal antibody 83.5 (West et al., 1986). The focus of 
my initial investigation was to characterize the mutation in 
HL250 with the help of biochemical and morphological 
techniques. We determined that HL250 is a fucosylation 
mutant that lacked cellular fucose when grown in the absence 
of fucose and that the defect is probably a result of a 
lesion detectable in vitro in the conversion pathway that 
forms GDP- fucose from GDP-mannose. 

Materials and Methods 
Materials 

GDP-[l- 3 H]mannose (9.1 Ci/mmol) was purchased from New 
England Nuclear and L-[ (5,6) - 3 H] -fucose (60 Ci/mmol) from 
American Radiochemical Corporation. KC1 and MgCl were 



20 
obtained from Mallinckrodt; formic acid from Fisher; ATP 
(disodium salt, catalog number A-5394), niacinamide, NAD + , 
NADPH, Trizma base, phenylmethylsulfonyl fluoride, Dowex-1 
(1x8, minus 400, chloride form), hexokinase, GDP-mannose, 
and Amberlite MB-3 were obtained from Sigma. ATP was stored 
frozen at a concentration of 500 mM in 1 mM Tris-HCl (pH 
7.4), resulting in a final pH of approximately 5.5. Dowex-1 
formate form was made as follows: 1) The column was washed 
with 1 M HC1 until pH of eluate was below 2 (as determined 
by pH paper). 2) The column was washed with water until pH 
was higher than 4.5. 3) Subseguently, the column was washed 
with 1 M NaOH until pH was higher than 13. 4) The column 
was washed with water as described in step 2. 5) The column 
was washed with 3 volumes of 1 M formic acid until pH of 
eluate was below 2. 6) Lastly, the column was washed with 
water as described in step 2 . This procedure was also 
followed for regeneration of column. 

Strains and Conditions of Growth and Development 

Dictyostelium discoideum amoebae were grown on HL-5, a 
medium that contains glucose, yeast extract, and proteose 
peptone (Loomis, 1971). The axenic strains Ax3 (from F. 
Rothman) and HL250 (from W.F. Loomis) were maintained by 
passage during logarithmic growth phase. Ax3 is the normal 
strain and HL250 is a mutant obtained from Ax3 by N-methyl- 
N' -nitro-N-nitrosoguanidine mutagenesis. For metabolic 



21 
labelling experiments, cells were grown for 4-6 doublings in 
8-20 jiCi/ml (0.10-0.26 vM) of [ 3 H]fucose in FM medium, a 
minimal defined medium that lacks fucose (Franke and Kessin, 
1977). When appropriate, the medium was supplemented with 
L-fucose (Sigma). For development, cells were plated in PDF 
buffer (20 mM KC1, 45 mM sodium phosphate, 6 mM MgS0 4 , pH 
5.8) on filters as previously described (West and Erdos, 
1988). 

Cell Lysis and Fractionation 

Logarithmically growing amoebas were harvested and 
washed in 50 mM Tris-HCl (pH 7.5) and resuspended to a 
concentration of 2xl0 8 cells/ml in the lysis buffer 
consisting of 0.25 M sucrose, 50 mM Tris-HCl buffer (pH 7.5) 
supplemented with 1 mM PMSF. All operations were carried 
out at 0-4 °C. Cells were immediately lysed by forced 
passage through a 5 ^m nuclepore polycarbonate filter, with 
pore diameter slightly smaller than the diameter of the 
cells (Das and Henderson, 1986). This method routinely 
yields more than 99% cell breakage. The lysate was 
centrifuged at 100k xg for 1 hour. The supernatant (S100) 
was made 2.5% (v/v) in glycerol by addition of 100% glycerol 
and either used immediately or saved at -80° without 
significant loss of activity for four weeks. 



22 

Localization by Immunofluorescence 

Prespore and spore cells were examined as described 
previously (West and Loomis, 1985; Gonzalez-Yanes et al., 
1989). The monoclonal antibodies utilized have been 
described previously elsewhere (West et al., 1986; Gonzalez- 
Yanes et al., 1989) . 

Determination of Fucose Content and Specific Activity 

The method is a modification of the protocol developed 
by Yurchenco and Atkinson (1975) for HeLa cells. Spores 
were harvested from sori not more than a day old and 
resuspended in water without washing, since it has been 
determined that a significant amount of spore coat protein 
may be lost after washing spores in water (West and Erdos, 
1990). Vegetative cells were grown in FM (in the presence 
or absence of extracellular L-fucose) for determination of 
sugar content. For determination of specific activity, 
cells were grown in FM media supplemented with [ H] fucose. 
After harvesting, cells were washed twice in PDF followed by 
EtOH precipitation. The ethanol supernatant was reextracted 
with EtOH and the resulting pellet pooled with the previous 
precipitate. EtOH was evaporated under a stream of air. 
Alternate methods such as TCA precipitation of the ethanol 
supernatant, did not significantly increase the yield. 
Samples were then hydrolyzed in a reacti-vial (Pierce) in 
0.1 N HC1 for 45 min at 100° on a heating block. 



23 
Macromolecules were EtOH precipitated and the remaining 
supernatant dried down, resuspended in water, desalted on an 
Amberlite MB-3 column and dried by vacuum centrifugation. 
The samples were redissolved in water and analyzed using a 
Dionex Bio-LC ion chromatograph by the method of Hardy et 
al. (1988). Standards and modifications to the 
chromatography procedure have been published elsewhere 
(Gonzalez-Yanes et al., 1989). Specific activity, when 
applicable, was determined by counting elution fractions and 
was expressed as radioactivity present in the fucose peak 
divided by the amount of fucose detected by the pulsed 
amperometric detector coupled to the HPLC, with reference to 
previously established calibration curves for L-fucose 
(Hardy et al., 1988). Greater than 95% of the eluted 
radioactivity was recovered from the column eluate at the 
elution position of fucose. 

Assay for Conversion of GDP-mannose to GDP-fucose 

The conversion assay was carried out essentially as in 
Ripka et al . (1986). In short, the standard assay mixture 
contained in a final volume of 1 ml 600-750 uq S100 protein, 
10 mM niacinamide, 5 mM ATP, 0.2 mM NAD + , 0.2 mM NADPH, 7.5 
}M GDP-[ 3 H]mannose (approximately 10 5 cpm) in 50 mM Tris- 
HCl, pH 7.5. After incubation at 37° the reaction was 
stopped with 50 pi of 2 N HC1, the reaction mixture boiled 
for 20 min and subsequently neutralized with 55 pi of 2 N 



24 
NaOH. Quantitation of the conversion of GDP-mannose to GDP- 
fucose was achieved by determining the amount of fucose 
present after acid hydrolysis. Free mannose, released from 
GDP-mannose, was phosphorylated by adding 4 units of 
hexokinase in the presence of 5 mM ATP and 5 mM MgCl 2 at 37° 
for 1 hour. Hexokinase catalyzes the transfer of one 
phosphate to C-6 of any acceptor hexose. Since fucose lacks 
a hydroxyl group at position C-6, it cannot be 
phosphorylated. Parallel controls with no enzyme were used 
to correct for losses. The mixture was passed over a Dowex- 
1 (formate) column (0.6 x 5 cm) and eluted with water. 1 ml 
fractions were collected an aliguot of 100 yl from the 
eluate was counted using ScintiVerse LC (Fisher); fucose- 
associated radioactivity usually eluted by the first 2 ml. 
Calculation of K and V was done by the Lineweaver-Burk 

m max 

double reciprocal plot (1/v vs. 1/[S]) as discussed by 
Henderson (1985). 

Results 
Phenotypic Description of a Fucosylation Mutant 
The normal strain Ax3 was mutagenized with 
nitrosoguanidine and surviving clones were screened with 
anti-SP96 antiserum (Loomis, 1987). One of the clones, 
HL250, was selected due to its inability to bind anti-SP96 
antiserum which recognizes carbohydrate and peptide 
epitopes. HL250 has been found to have a more permeable 



25 
spore coat, lower germination efficiency in older spores, 
and a longer doubling time when compared to the parental 
strain Ax3 (Gonzalez-Yanes et al., 1989; West et al., 
manuscript in preparation) . 

Absence of a fucose-dependent epitope . The failure of 
HL250 to react with anti-SP96 antiserum suggested that the 
mutant might be deficient in a form of protein 
glycosylation. This hypothesis was confirmed by finding 
that HL250 did not react at appreciable levels with the 
fucose-dependent monoclonal antibody (mAb) 83.5 (West et 
al., 1986; Gonzalez-Yanes et al., 1989). Spores from mutant 
and the parental normal strains were subjected to SDS-PAGE 
and Western blotting and probed with mAbs 83.5 and A6.2 
(West et al., 1986; Gonzalez-Yanes et al., 1989). The 
latter monoclonal is specific for SP96. Consistent with the 
supposition that glycosylation is affected in HL250, SP96 
was reduced in apparent molecular weight compared to SP96 
from Ax3 spores (West et al., 1986). The absence of the 
fucose-dependent epitope was further examined in developing 
cells and spores. Cells were plated for development and 
slugs dissociated by shearing in the presence of EDTA. 
Cells and spores were then processed for indirect 
immunofluorescence. Figure 2-1 shows the localization of 
SP96 in spores and prespore cells. Ax3 exhibits peripheral 
labelling of spores and a punctate labelling from prespore 



Figure 2-1. Localization of SP96 in prespore and spore cells. 

Slugs cells were dissociated, placed onto polylysine-coated 
glass slides, fixed, permeabilized, and processed for 
indirect immunofluorescence using mAbs 83.5 or A6.2. 



27 



PRESPORE CELLS 
83.5 A6-2 



SPORES 



83.5 



A6-2 



CO 
X 

< 



o 

CVJ 





28 
vesicles using both mAb. However, HL250 shows the same 
pattern only when A6.2 is used, in agreement with the 
results observed by Western blotting. The fact that 
labelling of spores with A6.2 is similar in both strains 
indicates that spore coat localization of SP96 is not 
affected by the mutation. 

Fucose content of normal and mutant strains . Epitope 
recognition by mAb 83.5 was inhibited by L-fucose (West et 
al., 1986), so the possibility of a defect in fucosylation 
in strain HL250 was investigated. Initially, the 
macromolecular fucose content of vegetative cells grown in 
fucose-free media, and of spores was investigated. Ethanol 
insoluble macromolecules were acid hydrolyzed, ethanol 
precipitated, and the supernatant deionized and 
chromatographed on an alkaline anion-exchange column 
equipped with a pulsed amperometric detector. When 
authentic [ 3 H] fucose was fractionated in this manner, more 
than 95% of the radioactivity eluted at the fucose position 
(not shown) . Fucose content was found to be negligible in 
the mutant, HL250, both in spores and vegetative cells when 
compared to the normal strain, Ax3 (table 2-1). However, 
when HL250 cells were grown in FM supplemented with 1 mM L- 
fucose, they possessed detectable amounts of fucose. 
Previous investigators have found by autoradiography that 
[ 3 H] fucose incorporation is highest in prespore cells 



29 



cn 



m 
u 

> 

•H 
+J 

(0 

P 
CD 

cn 
<D 
> 

c 
id 

cn 

id 

n 

o 
a 

CO 

p 

c 

4J 

I 

T3 
C 



o 

c 

o 

p 
c 
(1) 
p 
c 
o 
u 

CD 
CO 

o 
u 

fa 



I 
cn 

cd 

XI 
ITS 



tn 
o 

c 
c 

£ 



CD 
tn 
O 
u 

3 



<d 
cn 
o 
u 

3 



P 
CT 



tn 

c 
o 

•H 

p 

•H 
V 

c 
o 
u 

CD 

a 
> 

p 



cd 
u 



Ci 
•H 

p 
tn 



73 73 

C C 



•a T3 

c ts 



00 

rH 

+1 ^" 
t-t «* 
co • 
in o 



en tn 

CD CD 

U U 

o o 

01 01 



r-- «* n *r 

■ • • ■ 

OHHH 

+1 +1 +1 +1 

o <h tn m 

• • • • 

o r- en o 

m in m ^ 



GO 

o ■* • rn 

• • CO • 

in h +i h 
+i +i m +i 
io oo • o 

rn«>HC7> 

00 00 iH VD 



rn 



+1 
o 



(N O 

• • 

o m o 
+i r» +i 
in o in 



HIOON 



CD 

tn 
o 
u 

3 

I 



CD 
tn 

u 

3 

i 



s s s s 

fa pbi fa fa 



CD 
(0 

XI 
CD 
O 

e 

(0 



CD 
<tJ 
XI 
CD 

O 
£ 

(TJ 



o 




o 


in 




in 


tn cn 


m 


CM 


X J 


X 


t3 


< ac 


< 


53 



H S "O CD 

CD fa C CD 

P <0 U 

(0 y W P 



73 <fl <w 






c 

(0 

cu 

s 



CD 



* 9 



6-0 
CD 



tn 



t-i 
u 

01 

n ® CD 
2*0 4J 



tn 

01 
U (0 



T3 
CD 

c 

•H 

£ 

c 

CD 

P 
CD 




H 

a 



£ 



8*8 

5 CD 



M "0 <0 
Cn Cn, 

(0 01 



cn 



o n 

CD 
01 P 
CD CD 



01 

o cn 
cj <o 

3 
fa 



73 
CD 

tn 

01 CU CD 

73 > 

CD 

P 

u 

CD 



a 



3 
4-i 

in 5 

i 



y x 



as 



CD 

tn 
p 

3 

tn 

CD 
OS 



CD 



tn 



tn 

c 
o 

■H 

P 
(0 

c 



T3 

c <" 

■H -P 

CD • 
Cn £ 01 
C CD 73 

<HH O 

5 Q.£ CD 

o ap p 

a M 3 CD CD 
co cn oi S 73 



£ 



30 
compared to prestalk and vegetative cells, so higher levels 
of fucose would be expected in spores (Lam and Siu, 1981; 
Gregg and Karp, 1978). The levels of glucose and mannose 
were measured for comparison to determine if there was a 
difference in the amounts of other sugars in the mutant. 
None of the contents of the other sugars were reduced in the 
mutant when grown in FM. Thus it seems that the lesion in 
HL250 is selectively affecting fucose metabolism, relative 
to that of other sugars. This is consistent with the fact 
that fucosylation is usually a terminal modification of 
oligosaccharides, so its addition is not a prerequisite for 
the addition of other sugars (Kornfeld and Kornfeld, 1985). 

Characterization of the Mutant Lesion 

Earlier investigations have described mutants with a 
fucose minus phenotype that are the result of a defect in 
GDP-fucose biosynthesis. The main source of GDP-fucose is 
the conversion pathway of GDP-mannose to GDP-fucose 
(Yurchenco et al., 1978; Flowers, 1985). It consists of the 
reactions presented in figure 2-2. Alternatively, synthesis 
of GDP-fucose by the fucose salvage pathway can occur in the 
presence of extracellular L-fucose (figure 2-2), allowing 
cells defective in the conversion pathway to phenotypically 
revert (Ripka and Stanley, 1986; Reitman et al., 1980). 
HL250 cells grown and developed in the presence of 1 mM L- 
fucose reexpressed the fucose epitope (Gonzalez-Yanes et 



Figure 2-2. Biosynthesis of GDP-fucose. 

Diagrams of the conversion pathway elucidated in bacteria 
and mammalian cells (adapted from Flowers, 1981) and the 
salvage pathway as described in HeLa cells (adapted from 
Yurchenko et al., 1978). 



32 




3,5-epimerttc 

> ° 



0-6DP 




■— O-GDP 



OH H 



A CONVERSION PATHWAY 





'""» v 1/ CHj 

nn«e \ H 



•Jl 



0- P 



-0 M 



pyrophosphoryliw I / tm \ 




•— O-GDP 



33 
al., 1989), and vegetative cells grown in 1 mM L-fucose had 
detectable amounts of macromolecular-associated fucose 
(table 2-2). These results suggested that HL250 had a 
normal salvage pathway, but a defect in the GDP-mannose to 
GDP-fucose conversion pathway. 

In vitro conversion of GDP-mannose to GDP-fucose . 
Conversion of GDP-mannose to GDP-fucose has been reported in 
bacteria, in higher plant cells, and mammalian cells 
(Kornfeld and Ginsburg, 1966; Liao and Barber, 1971; Ripka 
et al., 1986). It was assumed that Dictyostelium would 
share this ability with other species, so I assayed if cell 
extracts in vitro were able to convert GDP-mannose to GDP- 
fucose. High speed supernatants from Ax3 and HL250 were 
assayed in vitro for their ability to convert GDP- 
[ 3 H]mannose into GDP- [ 3 H] fucose. Cells were homogenized and 
a 100k x g soluble fraction (S100) assayed as described by 
Ripka et al. (1986) and the effects of time, varying protein 
and GDP-mannose concentrations were examined. Table 2-2 
shows that the conversion activity in Ax3 was proportional 
to the time of incubation. In contrast, mutant extracts 
showed negligible activity. Ax3 and HL250 cytosols were 
mixed to determine if a soluble inhibitor of GDP-mannose to 
GDP-fucose conversion activity was present. When equal 
amounts of extracts were mixed, activity was commensurate 
with the Ax3 contribution (table 2-2) indicating HL250 does 



34 



Table 2-2. 
GDP-fucose. 



time (mln) 



7.5 
15 
30 
90 



Effect of time on conversion of GDP-mannose to 
nmol fucose/mq protein 



Ax 3 

0.57 
2.5 
2.9 
9.2 



HL250 




0.15 




Ax3+HL250 (0.5:0.5) 

0.21 
1.3 
2.2 
4.3 



Protein (600 pg total) from a 100,000 xg supernatant of a 
vegetative cell-free extract was assayed for ability to 
convert GDP-[ 14 C]mannose (7.5 yM initial concentration) to GDP- 
[ 14 C]fucose; data are the result of the average of two 
determinations 



35 
not contain an inhibitor for the activity. Conversion was 
linear with respect to protein through 800 jig (figure 2-3, 
panel A) . In agreement with the results from the time 
dependence experiment, HL250 expressed less than 1% of the 
activity possessed by Ax3 at all concentrations of protein 
assayed (figure 2-3, panel A). Conversion by Ax3 S100 was 
also dependent on the amount of GDP-mannose present while 
the activity present in HL250 was insignificant (figure 2-3, 
panel B) . The Ax3 GDP-mannose to GDP-fucose conversion 
activity showed an apparent K m of 14.1 uM and V^ of 18.3 
nmol fucose/mg protein/30 min (fig. 2-3, panel C) . Previous 
reported values for the apparent K m of the conversion 
pathway range from 2 uM in CHO cells (Ripka et al., 1986) to 
160 uM in the higher plant Phaseolus vulgaris (Liao and 
Barber, 1971). 

The conversion activity is absent in mutant extracts in 
vitro, if absent in vivo, this would explain the lack of 
fucose in living cells and the correction by exogenous 
fucose. Taken together, all these results point to the 
conversion pathway as the site of the lesion in HL250. 
Furthermore, the defect seems to be in the first step of the 
conversion of GDP-mannose to GDP-fucose because no 
radioactivity was recovered after hexokinase treatment. If 
the 4, 6 -dehydratase was active, the product would have 
eluted from the ion-exchange column after hydrolysis and 
phosphorylation by hexokinase (see figure 2-2). 



Figure 2-3. Conversion of GDP-mannose to GDP-fucose by normal 
and mutant strains. Closed circles, Ax3; open circles, 
HL250. Results are the average of two determinations. 

Panel A. Effect of protein concentration on conversion. 30 
min. assay, 7.5 jiM GDP-[ 3 H]mannose. 

Panel B. Effect of GDP-mannose concentration on conversion. 
30 min assay, 750 jig protein for each strain. 

Panel C. Apparent Michael is constant for GDP-[ H]mannose, 
determined for Ax3 conversion. Apparent K m was determined 
to be 14.1 jiM and apparent V max 18.3 nmol/mg protein/30 min 
by the Lineweaver-Burk double reciprocal plot method. 



37 



E 
o 

CO 

w 

8 



o 

E 




200 400 600 

ng protein 



800 



I 

o 

CO 

's 

o 

i_ 

Q. 
O) 

E 

a5 
t/5 
o 
o 

o 

E 

c 





B 






10- 








5- 






c>i 


o c! 


5 r-<J=T ^V 


u 

i — ' — i — > — 





10 15 20 

GDP-mannose (^.M) 



25 



u.o - 


c 




0.2- 






0.1 - 






0.0- 


' < 


I ■ 1 ' 1 ' 1 — ' 



■0.09 



-0.00 



0.09 
1/[S] 



0.18 



0.27 



38 
Specific activity of fucose pools . A lesion in the 
GDP-mannose to GDP- fucose conversion pathway would render 
the mutant defective in macromolecular fucosylation when 
grown in the absence of fucose, as was shown earlier. Such 
a scenario would require that the GDP-fucose inside the cell 
be derived from the salvage pathway fed exclusively by 
fucose from the extracellular media. In the case of the 
Ax3, however, there would be a contribution of GDP-fucose 
derived from the conversion pathway. If the mutation in 
HL250 was indeed in the GDP-mannose to GDP-fucose 
conversion, macromolecular fucose of cells grown in the 
presence of L-[ 3 H] fucose would have the same specific 
activity as the fucose present in the media. To test this 
hypothesis, mutant and normal cells were grown in fucose- 
free defined media supplemented with [ H] fucose. Whole cell 
preparations were then assayed for fucose content as 
described above and the specific activity expressed as the 
radioactivity present in the fucose peak divided by the 
amount of fucose detected. As expected, the macromolecular 
pool of the mutant had essentially the same specific 
activity as the fucose in the medium. In contrast, the 
specific activity in Ax3 was diluted approximately 400-fold 
compared to the medium (table 2-3). These results confirmed 
that HL250 derived its intracellular fucose from the salvage 
pathway. Meanwhile it appears that in Ax3 the contribution 



39 



Table 2-3. Specific activities of fucose. 

cpm/nmol fucose 
strain fucose concentration medium macromolecular 

HL250 50 pM 1.1x10* 9.5x10^ 

Ax3 0.1|iM 1.8x10 4.7x10 



Cells were grown for 3 days in FM media in the presence of 
6xl0 6 dpm/ml of [ 3 H] fucose supplemented with non-radioactive 
fucose to yield the noted concentration of fucose in the 
media. Specific activity was determined as described in 
Materials and Methods. 



40 
of fucose by the salvage pathway is minimal, 1/400 of the 
total content. These studies agree with earlier reports on 
fucose metabolism/ where the majority of fucose in mammalian 
cells is derived from the conversion of GDP-mannose to GDP- 
fucose (Yurchenko et al. f 1978). 

Discussion 

HL250 failed to express a fucose-dependent epitope 
recognized by the mAb 83.5. However, it expressed SP96, one 
of the polypeptides that bears the carbohydrate epitope 
recognized by 83.5. These results were reproduced by 
Western blot (Gonzalez-Yanes et al., 1989) and indirect 
immunofluorescence. Interestingly, the immunofluorescence 
microscopy studies showed that the mutant is not defective 
in its ability to package SP96 in vesicles or in the 
targeting of the glycoprotein to the spore coat. Similar 
results were reported for other proteins in another 
Dictyostelium glycosylation mutant (Aparicio et al., 1990; 
West and Loomis, 1985). Measurements of fucose content of 
cells and spores demonstrated that the mutant contained 
almost undetectable amounts of fucose, in contrast to Ax3 
which contained macromolecular-associated fucose in both 
cell types. 

Once HL250 was identified as having a mutation that 
resulted in decreased macromolecular fucose, I tried to 
identify the nature of the lesion. It was speculated that 



41 
the mutant HL250 may have a defect in (1) fucosyltransf erase 
activities, (2) endogenous acceptors for 
fucosyltransf erases, (3) transport of GDP-fucose into 
microsomal vesicles, and/or (4) synthesis of GDP-fucose. In 
vitro microsomal extracts of normal and mutant cells were 
active in the transfer of [ 14 C]fucose from GDP- [ 14 C] fucose to 
endogenous acceptors and the activity was latent (see 
Chapter IV), so it was reasoned that fucosyltransferases may 
be normal and probably the uptake of GDP-fucose by vesicles 
was not impaired, so the lesion might be at another point in 
the fucosylation pathway. Since fucose is normally added as 
a terminal modification, the fucose minus phenotype could be 
the result of a lack of formation of acceptors for the 
fucosyltransferases (Stanley, 1984; Hirschberg et al . , 
1982). HL250 expresses levels comparable with Ax3 of other 
carbohydrate epitopes and has normal neutral monosaccharide 
composition (Gonzalez-Yanes et al., 1989; West et al., 
1986), for these reasons it was speculated that the defect 
was not in an earlier step of glycosylation but it involved 
the fucosylation pathway directly. 

The glycosylation defect in HL250 appears to result 
from an inability to produce GDP-fucose. I have found that 
the GDP-mannose to GDP-fucose conversion activity in vitro 
is reduced to undetectable levels in mutant cell extracts. 
The fact that there is partial rescue when the cells are 
grown in the presence of fucose, suggests that the cells are 



42 
producing GDP-fucose via the salvage pathway and that the 
rest of the fucosylation machinery is probably normal. 
Earlier studies have reported the phenotypic reversion of 
mammalian mutants with a defective GDP-mannose to GDP-fucose 
conversion pathway when the cells were supplied with 
extracellular fucose (Ripka and Stanley, 1986; Reitman et 
al., 1980). However, Ripka and Stanley (1986) used the 
recovery of lectin sensitivity as a marker for phenotypic 
reversion, but did not report measuring the fucose content 
of the cells or show the data for lectin binding compared to 
the parental strain. Reitman et al. (1980) showed that a 
mouse lymphoma cell line which has a defect in the 
conversion of GDP-mannose to GDP-fucose was defective in pea 
lectin binding compared to the parental cell line. The 
ability to bind pea lectin was restored to wild type 
parental cell line levels after culturing in 10 mM fucose. 
Fucose is an important determinant in the carbohydrate- 
binding specificity of pea lectin (Kornfeld et al., 1981). 
It is important to note that the mutant mouse lymphoma cell 
line had approximately one fifth the amount of fucose and 
one third the number of high affinity lectin-binding sites 
as the parental line, indicating that mutant cells were 
salvaging fucose from the medium, or that the mutation was 
only partial. The evaluation for phenotypic reversion of 
HL250 is more rigorous since it demands the expression of a 



43 
carbohydrate epitope and measures total levels of fucose 
from a cell that was grown in fucose-free media. 

The specific activity of the medium was compared to the 
specific activity of the intracellular macromolecular 
fucose. Consistent with a lesion in the GDP-mannose to GDP- 
fucose conversion pathway, the mutant cells relied on 
extracellular fucose as their only fucose source. In 
contrast, extracellular fucose only contributed to a small 
fraction of the total Ax3 fucose pool. This is useful 
because it means that radioactivity from cells grown in 
[ 3 H] fucose can be used as a direct measure of fucosylation 
in HL250. 

In conclusion, even though HL250 has a severe 
glycosylation lesion which renders it unable to carry out 
fucosylation when grown in the absence of fucose, the strain 
is able to grow, develop, and form spores. To my knowledge, 
there are no previous reports in the literature of 
eukaryotic cells defective in the GDP-mannose to GDP-fucose 
conversion pathway that can survive in fucose-free media. 
The fucosylation mutants reported are cell lines that have a 
functional salvage pathway maintained in culture in the 
presence of animal serum, so they can synthesize GDP-fucose 
from the fucose present in the cell culture media (Ripka and 
Stanley, 1986; Reitman et al., 1980). For this reason, 
these investigators were unable to totally deprive the 
mutants of fucose, as I am able to do with Dictyostelium. 



44 
Other lower eukaryotes, such as yeast, do not carry out 
fucosylation (Kukuruzinska et al., 1987) so the existence of 
a Dictyostelium fucosylation mutant could be very important 
to study fucosylation. In any event, HL250 has already 
served as a very useful tool in which to study fucosylation 
events, as will be evident in the following chapters. 



CHAPTER III 
IDENTIFICATION OF A CYTOSOLIC FUCOPROTEIN 



Introduction 
There is some evidence that fucoconjugates are present 
in the nucleus and cytosol (Hart et al., 1989a; Chapter I). 
However, virtually nothing is known about the structure or 
biosynthesis of these fucosylated macromolecules . Most 
studies have limited themselves to reporting the existence 
of evidence for nuclear or cytoplasmic glycoproteins, but 
have not gone further to characterize the sugar-peptide 
linkage or compare it with material derived from the 
secretory pathway. As discussed in Chapter II, there is a 
conditional fucosylation mutant, HL250, that can be readily 
labelled when grown in radioactive fucose. Using this 
strain, I have identified a fucoprotein that fractionated 
with the cytosol and appeared to be the major fucosylated 
species in the cytosol. The oligosaccharide-peptide linkage 
was characterized and the fucoprotein was compared with 
fucosylated material derived from vesicles, and 
differentiated based upon several criteria. 



45 



46 
Materials and Methods 
Materials 

L-[ (5,6)- 3 H]-fucose (60 Ci/mmol) was obtained from 
American Radiochemical Corporation and D-[ (2, 3)- H]mannose 
(24 Ci/mmol) from New England Nuclear. TS-1 was purchased 
from Research Product International; POPOP was from 
Mallinckrodt; SDS, leupeptin, aprotinin, phenyl 
methylsulfonyl fluoride, Triton X-100, 

dimethyldichlorosilane, MES, all nitro-phenyl substrates, 
mannose-6-phosphate, bovine serum albumin (fraction V), blue 
dextran, bromo phenol blue, and trypsin were from Sigma; 
glycine, benzene, ammonium acetate, PPO, and toluene were 
from Fisher. The concentrations of Triton X-100 and NP-40 
are expressed as v/v, all others are expressed as w/v, 
unless specified otherwise. 

Strains and Conditions of Growth 

Dictyostelium discoideum strains Ax3 (from S. Free) and 
HL250 (from W.F. Loomis) were grown on HL-5, a complete 
medium that contains glucose, yeast extract, and proteose 
peptone (Loomis, 1971). Ax3 is the normal strain and HL250 
is a mutant obtained from Ax3 by N-methyl-N' -nitro-N- 
nitrosoguanidine mutagenesis (Loomis, 1987). HL250 lacks 
the enzyme activity that converts GDP-mannose into GDP- 
fucose which results in a lack of cell fucose (Gonzalez- 
Yanes et al., 1989; also see Chapter II). In all 



47 
experiments cells were collected at the logarithmic growth 
phase, with a cell density of 1 to 9 x 10 6 cells/ml. For 
metabolic labelling experiments, cells were grown for 4-6 
doublings in 2-20 jiCi/ml (0.03-0.26 ^M) of L-[ 3 H]-fucose in 
FM medium, a minimal defined medium that lacks fucose 
(Franke and Kessin, 1977). 

Cell Lysis and Fractionation 

Logarithmically growing amoebae were harvested and 
washed in 50 mM MES (pH 7.4) and resuspended to a 
concentration of 2xl0 8 cells/ml in the lysis buffer 
consisting of 0.25 M sucrose, 50 mM MES buffer (pH 7.4) 
supplemented with the protease inhibitors leupeptin ( 10 
|ig/ml), aprotinin (10 vq/ml) , and PMSF (1 mM) . When 
specified, cells were fractionated in the presence of a 
comprehensive cocktail of protease inhibitors which have 
been developed for the isolation of various proteolitically 
sensitive proteins in Dictyostelium (Goodloe-Holland & Luna, 
1987; Stone, et al., 1987). All steps were carried out at 
0-4 °C. At once, the cells were gently lysed by forced 
passage through a 5 jim nuclepore polycarbonate filter, with 
a pore diameter slightly smaller than the diameter of the 
cells (Das and Henderson, 1986). This method routinely 
yielded more than 99% cell breakage as assessed by contrast 
phase microscopy. The lysate was clarified from unbroken 
cells and nuclei by a 2k xg centrifugation for 5 min, then 



48 
it was centrifuged at 100k xg for 1 hour, unless otherwise 
specified. The pellet (P100) was resuspended in lysis 
buffer by pipetting to the same volume as the supernatant 
(S100). For lysing vesicles, the P100 was sonicated using a 
Branson Sonifier Cell Disrupter 185. 

Slug cells were plated for development as described 
earlier (West and Erdos, 1988), and harvested in buffer of 
Berger and Clark (as described in West and Brownstein, 1987) 
supplemented with 20 mM EDTA. Cells were dissociated in 
this buffer by passing 20 times through a long, 9 inches, 
pasteur pipette, followed by passing 20 times through a 23- 
gauge needle. EDTA was washed by resuspending cells in 50 
mM MES, pH 7.4, titrated with NaOH. Cells were resuspended 
in lysis buffer and immediately lysed by passage through a 3 
/L/m nuclepore polycarbonate filter (Das and Henderson, 1986). 
Cell lysates were then treated as described above for 
vegetative cells. 

Gel Electrophoresis and Western Blotting 

SDS-PAGE was carried out under reducing conditions 
essentially as described in West & Loomis (1985). Samples 
were resolved by 7-20% acrylamide linear gradient gels or 
15% acrylamide gels and, initially, molecular weight 
assigned using low MW markers kit (Sigma). In later 
experiments, trypsin was used as a molecular weight marker. 
Following electrophoresis, the gels were cut immediately 



49 
and/or stained and destained and then cut into either 2.2 mm 
or 0.5 cm slices. Gel pieces were shaken and swollen 
overnight in a scintillation cocktail composed of 111.1 ml 
of tissue solubilizer (TS-1), 6.0 g PPO, 0.15 g POPOP, and 
20 ml dH to 1 1 of toluene. Gel slices were counted and 
recounted until dpm were determined to be stable, usually 1- 
2 days later. For gel-purified material, the sample was run 
in a 7-20% linear gradient gel and the 21 kD area 
(approximately 1 cm below trypsin) cut out and electroeluted 
overnight using a Bio-Rad electroeluter following 
manufacturer's directions, except the Laemmli 
electrophoresis buffer used for SDS-PAGE (West and Loomis, 
1985) was used instead of the recommended volatile buffer to 
avoid alkaline hydrolysis. Western blotting was carried out 
as previously described (West and Loomis, 1985). 

Partial Purification of FP21 by Anion Exchange 
Chromatography 

In preliminary studies, FP21 was partially purified by 

fractionation of metabolically labelled S100 fraction on a 

TSK DEAE-5PW 8 x 75-mm HPLC anion-exchange column (LKB) 

preequilibrated with 10 mM NH 4 Ac, pH 7.0. Sample was 

dialyzed against 2 1 of 10 mM NH 4 Ac, pH 7.0, for several 

hours, and clarified by centrifugation at 10k x g for 10 min 

prior to injection. Protein was eluted using an increasing 

linear gradient (10 mM to 1 M NH 4 Ac, pH 7.0) for 40 min at a 

rate of 0.75 ml per min, and the majority of FP21 was found 



50 
to elute at 0.5 M input buffer concentration. Fractions 
were analyzed by SDS-PAGE and counting of the gel slices. 

Protein Concentration Assay 

The Bio-Rad protein assay was used for determination of 
protein concentration, and bovine serum albumin used as 
standard. 

Enzyme Assays 

a-glucosidase-2 assays contained 100-300 yq protein, 
8.6 mM p-nitrophenyl-ot-D-glucoside, 0.1% Triton X-100, in 21 
mM citrate-phosphate buffer (pH 7.5) at 37° (Borts and 
Dimond, 1981). Reaction was stopped after 1 hr by addition 
of Na 2 C0 3 to a concentration of 0.5 M and the absorbance 
read at 420 nm. Glucose-6-phosphatase and mannose-6- 
phosphatase were measured by release of phosphate from 
mannose-6 -phosphate, which has been previously shown to be a 
suitable substrate for both enzymes (Arion et al., 1976). 
Reaction mixtures contained 100-300 vg protein, 1 mM MgCl 2 , 
2 mM mannose-6-phosphate, 0.1% Triton X-100 in 10 mM MES 
(titrated with NaOH to a pH of 7.4) in a volume of 200 jil. 
After 20 min incubations at 30° reactions were stopped by 
adding 200 jul 20% ice-cold TCA (Snider et al., 1980). Tubes 
were centrifuged at 14k rpm, for 10 min in an Eppendorf 
table top microfuge and aliquots of the supernatants were 
assayed for P 1 by the method of Chen et al. (1956). Acid 



51 
phosphatase was assayed as described in McMahon et al . 
(1977) except that Triton X-100 was included at a 
concentration of 0.1% and absorbance was measured at 420 nm, 
instead of 400 nm. 

PNGase F Digestion 

Gel-purified FP21 (3000-8000 dpm) was boiled in 0.5% 
SDS in water for 3 min. For digestion, the protocol of 
Tarentino et al . (1985) was followed. The sample was 
incubated in 104 mM sodium phosphate, pH 8.6, 10 mM EDTA, 10 
mM 1,10-phenanthroline (stock solution of 100 mM in 
methanol), 2% NP-40, 0.21% SDS, and 20 U/ml PNGase F 
(Boehringer-Mannheim) in 300 ^1 for 22 h at 37°. Fetuin (B- 
grade, Calbiochem) and RNAse B (Sigma) were treated 
identically as controls and digestion was quantitative, or 
nearly quantitative, as determined by shifts in molecular 
weight in SDS-PAGE. In one trial FP21 was digested with 
trypsin prior to PNGase F digestion by incubating gel 
purified FP21 in 0.8 mg/ml trypsin, 1 mM CaCl 2 for 1 hr at 
37°. The reaction was stopped by boiling in the presence of 
1 mM PMSF and 10 luq/ml aprotinin for 3 min. 

Pronase Digestion 

Glycopeptides were prepared by exhaustive pronase 
digestion as described (Ivatt et al, 1984). In short, after 
gel purification and electroelution, the samples were dried 



52 
down and resuspended in water to a volume of 200 ill 
containing at least 5xl0 3 dpm. In other experiments, 200 vl 
of the entire P100 fraction were left intact or made 0.1% 
Triton X-100. 200 jil of freshly dissolved 1% pronase 
(CalBiochem) in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl 2 , were 
added at 0, 24, and 48 hours. Incubation was at 50° and a 
few drops of toluene were added to prevent microbial growth. 
At 72 hours the reaction was stopped by incubating in a 
boiling water bath for 3 min. 

Oligosaccharide Release by Alkaline-Borohydride Treatment 

The oligosaccharide in FP21 was released by mild 
alkaline hydrolysis under reducing conditions, also known as 
fl-elimination. To approximately 5xl0 3 dpm of gel purified 
protein, freshly made NaOH and NaBH 4 concentrated solutions 
were added in that order to yield a final concentration of 
0.1 M and 1 M, respectively. Samples were incubated for 15 
hours in a water bath at 45°. The reaction was stopped by 
the addition of acetic acid to a final concentration of 1 M. 
The samples were dried down by vacuum centrifugation, 
resuspended once in 1 ml 100 mM HAc, dried again, and 
resuspended twice in methanol and stored dry at -80° until 
ready to use. 

The oligosaccharide was also released by strong 
alkaline-borohydride treatment. The method is that of Zinn 
et al. (1978) and very similar to the procedure for R- 



53 
elimination, with some exceptions: NaOH and NaBH 4 were 
present at a final concentration of 1 and 4 M, respectively, 
and samples were incubated at 80° for 24 h. Reaction was 
terminated by diluting the sample twofold with water and 
adding acetic acid to a final concentration of 4 M. Borate 
salts were removed by methanol evaporation as described 
above . 

P-4 Column Fractionation 

Dry samples from fi-elimination and strong alkaline- 
borohydride treatment were resuspended in 800 yl of 50 mM 
pyridinium acetate (pH 5.5). Pyridinium acetate was made in 
the hood by mixing in water, to a final volume of 2 liters, 
8.06 ml pyridine, 2.17 ml glacial acetic acid. After the 
previous reagents were dissolved, 0.4 g of sodium azide was 
added. The solution had a final pH of approximately 5.5. 
The buffer was degassed and stored under chloroform 
atmosphere. Samples from pronase digestion were centrifuged 
for 5 min at 14k rpm on a Eppendorf table top microfuge and 
the supernatant taken for P-4 chromatography. 
Oligosaccharides and glycopeptides were fractionated in a 
0.9 cm x 1 m BioGel P-4 column (-400 mesh) equilibrated with 
50 mM pyridinium acetate (pH 5.5) as the mobile phase. 
Prior to pouring, the column was acid washed overnight and 
siliconized by coating with 1% (v/v) dimethyldichlorosilane 
in benzene for 10 min. The column was calibrated with 



54 
glucose oligomers used as standards (Yamashita et al., 1982) 
that were derived from a dextran hydrolysate which was 
reduced with NaB 3 H 4 (kindly provided by J. Baezinger) . 
Twelve drop fractions (approximately 250 /il) were collected 
and counted using ScintiVerse LC (Fisher Scientific). All 
runs were performed at 37°. Recovery varied somewhat 
between runs, but it was between 30-80% of the loaded 
radioactivity. The void volume (Vo) was determined with 
blue dextran (at a concentration of 0.2%), and the inclusion 
volume (Vi) with either bromo phenol blue (0.2%) or 
[ 3 H]mannose (approximately 2,000 dpm) . The relative elution 
coefficient (Rev) for each component was determined from the 
elution volume (Ve) : Rev = (Ve-Vo) /(Vi-Vo) . 

Results 
Analysis of Cellular Fucoproteins by SDS-PAGE 

HL250 amoebae were grown in minimal defined media 
supplemented with L-[ 3 H]fucose. During logarithmic growth 
phase, cells were harvested, washed, and resuspended in 0.25 
M sucrose buffer supplemented with protease inhibitors. 
Immediately, the cells were lysed and the lysate was 
clarified from unbroken cells and nuclei by centrifugation 
at 2k x g for 5 min. The resulting supernatant from this 
spin was centrifuged at 100k x g for 1 hour. Both fractions 
were analyzed by SDS-PAGE and the gels stained, cut into 2.2 
mm slices, and counted. While total protein distributed in 



55 
a ratio of almost 1:1 (P100:S100), the specific activity 
(expressed as dpm/mg of protein) distributed roughly in a 
6:1 ratio (P100:S100) (table 3-1). The majority of the 
radioactivity fractionated with the P100. However, the 
amounts of radioactivity recovered in the S100 were 
unexpected; 36% of the radioactivity in the S100 (this value 
varied in individual experiments from 30-68%) migrated as 
one peak at the 21 kD position, slightly ahead of trypsin, 
which was used as a molecular weight marker. On the other 
hand, the P100 showed two main broad peaks, one at 10-25 kD 
and another at 58-84 kD with 10% of the total radioactivity 
in the P100 migrating at the 21 kD level (figure 3-1). The 
proteinaceous nature of the S100 fucoprotein was confirmed 
by digestion with the proteases trypsin and pronase with 
guantitative recovery of radioactivity at lower MW positions 
in unfixed gels (results from pronase digestion shown in 
figure 3-2). The S100 fucosylated protein has been called 
FP21 for fucoprotein of 21 kD molecular weight. 

The abundance of FP21 was estimated based on the 
specific activity of fucose and determined to be 10 3 
molecules in FP21, assuming one fucose molecule per molecule 
of FP21. Based on the dilution of fucose specific activity 
in Ax3 (from table 2-3, Chapter II), there would be 4 x 10 5 
molecules in Ax3. If there is one fucose per copy of FP21, 
and all FP21 molecules are fucosylated (evidence for 
guantitative FP21 fucosylation in Ax3 will be presented in 



56 



Table 3-1. Distribution of protein and radioactivity in S100 
and P100 fractions. 

S100 P100 

total protein 

(equivalents) 1 0.95 

specific activity 

3 days 14 dpm//jg 88 dpm/£/g 

5 days 13 dpm/^g 225 dpm//jg 



HL250 amoebae were grown in the presence of 2 /iCi/ml of 
[ 3 H]fucose in FM media for the indicated period of time. Data 
are from one representative experiment. Cells were harvested, 
filter lysed, and fractionated into an S100 and P100. 



Figure 3-1. Incorporation of [ 3 H]fucose into macromolecular 
species of the S100 and P100. 

HL250 amoebae were metabolically labelled with 2 jjCi/ml of 
[ 3 H]fucose, lysed, fractionated into an S100 and P100, and 
subjected to 7-20% linear gradient SDS-PAGE; the gel was 
sliced into 2.2 mm pieces and counted. 100 vq of protein 
were electrophoresed for the S100 and P100, respectively. 
Open circles, P100; closed circles, S100; arrow, migration 
of trypsin. 



58 



D 



o 
o 

CO 



2000 



1600- 



1200- 



800 



400 




20 30 40 

gel slice 



600 



-500 



-400 



-300 



-200 



-100 



Q 



o 
o 



Figure 3-2. Proteinaceous nature of FP21. 

Approximately 1,500 dpm of metabolically [ 3 H]fucose-labelled 
FP21 from HL250 was gel purified, as described in Materials 
and Methods, and subjected to either a mock or pronase 
digestion. Resulting digests were electrophoresed on a 15% 
SDS polyacrylamide gel, which was then sliced into 0.5 cm 
pieces and counted. Open circles, FP21; closed circles, 
FP21 digested with pronase. 



60 



160 



120 



0- 80- 
Q 



40 



trypsin dye front 




r-+ 



2 4 



6 



—I— 
10 



— T— 
12 



14 16 



gel slice 



61 
Chapter IV), and the number of copies of FP21 per cell is 
not affected by the mutation, then there may be a maximum of 
4 x 10 5 copies of FP21 per cell. 

Evidence that FP21 is Endogenous to the Cytosol 

Fucosylation has been shown to occur in the Golgi 
apparatus in other organisms (Hirschberg and Snider, 1987; 
Kornfeld and Kornfeld, 1985), so I assessed the possibility 
that FP21 was a lumenal microsomal protein that leaked 
during the P100 and S100 isolation procedure. To guard the 
P100 from chemical lysis, the vesicles were prepared in a 
cocktail of protease inhibitors that contained additional 
inhibitors from those utilized in the standard fractionation 
protocol, and which have been developed for the isolation of 
various proteolitically sensitive proteins in Dictyostelium 
(Goodloe-Holland & Luna, 1987; Stone, et al . , 1987). To 
address the possibility of mechanical disruption of the 
vesicles, the P100 was disrupted by sonication and 
recentrifuged at 100k x g for 1 h. Approximately 11% of the 
radioactivity was released and the supernatant of this 
centrifugation was analyzed by SDS-PAGE as described above 
(figure 3-3). Although several radioactive peaks were 
present in the supernatant of the second centrifugation, the 
radioactivity profile was different from the S100 suggesting 
that FP21 is not a protein released by disruption of the 
vesicle fraction. Although radioactivity which comigrated 



Figure 3-3. Comparison of S100 and releasable P100 components. 

HL250 amoebae were metabolically labelled with 2 /jCi/ml of 
[ 3 H]fucose, lysed, fractionated into an S100 and P100. The 
P100 was sonicated and recentrifuged. After centrif ligation, 
the resulting supernatant was examined by slicing 7-20% 
linear gradient SDS-PAGE into 2.2 mm and counting the gel 
pieces (panel B) . Included for comparison, is the profile 
from the S100 radiolabeled species from the same 
preparation run on the same gel (panel A) . 50 ^g of protein 
were electrophoresed for the S100 and P100, respectively. 
Arrow, migration of trypsin. 



63 



120 




a. 
Q 



200 



150 - 



100 - 



50 - 




1 



20 30 40 

gel slice 



50 



64 
with FP21 was observed in the PlOO-derived supernatant, this 
material was a minority of the radioactivity released, and 
probably reflected the general heterogeneity of the P100 
vesicle contents. Less than 1% of the total cell 
radioactivity that migrated at the 21 kD molecular weight 
position was released from the P100 by sonication, 
indicating that the remaining FP21 is recovered in the S100. 

In a different approach, P100 from in vivo [ H]fucose 
labelled cells was mixed with unlabelled post-nuclear 
supernatant and recentrifuged (table 3-2). More than 98% of 
the radioactivity sedimented with the P100, suggesting that 
once associated with the P100, radioactivity is not lost, 
unless vesicles are purposely disrupted, as in sonication. 

The enzymes a-glucosidase-2, glucose-6-phosphatase, and 
acid phosphatase have been used as markers of the 
endoplasmic reticulum, Golgi apparatus and endoplasmic 
reticulum, and lysosomes, respectively (Borts and Dimond, 
1981; McMahon et al., 1977). To examine the distribution of 
these enzyme markers in the high speed fractions, HL250 
amoebae were harvested, homogenized, fractionated into an 
S100 and P100 and assayed for activity of the different 
marker enzymes. As seen in table 3-3, the majority of the 
activity was recovered in the P100, suggesting minimal 
contamination of the S100 by vesicles containing these 
enzymes. Less than 7% of the total a-glucosidase-2 activity 



65 



Table 3-2. Radioactivity recovered in the second S100 after 
different P100 treatments. 

condition % radioactivity recovered 

untreated 2.2% 

sonicated 11.3% 

mixed* 1.4% 



Cells were labelled in vivo by growing in 2 fuCi/ml of 
[ 3 H]fucose in FM media for 3 days, lysed, and fractionated 
into an S100 and P100. The P100 was then subjected to 
different treatments and recentrifuged at 100k x g for 1 hr. 
The S100 from this second centrifugation was analysed for 
radioactivity. *P100 from metabolically labelled cells was 
mixed with unlabelled post-nuclear supernatant from cells 
grown in FM media and recentrifuged at 100k x g for 1 hr. 



66 



Table 3-3. Distribution of markers among S100 and P100 
fractions. 

S100 P100 

a-glucosidase-2 6.5% 93.5% 

acid phosphatase 12.8% 87.2% 

glucose-6-phosphatase n.d. 100% 



HL250 amoebae grown in HL-5 were fractionated into S100 and 
P100, and assayed for activity as described in Materials and 
Methods. Activity expressed as percentage of total activity 
detected in both fractions, n.d., not detectable. 



67 
was found in the S100; mannose-6-phosphatase was only 
detectable in the P100. More than 87% of the activity of 
the lysosomal enzyme acid phosphatase was detected in the 
P100. In Dictyostelium, this enzyme has been shown to be a 
soluble lumenal lysosomal protein (Dimond, et.el., 1981). 

Further evidence that P100 vesicles are stable, closed 
structures comes from earlier studies from the laboratory. 
Prespore proteins SP75 and SP96 sediment at 100k x g unless 
cells are sonicated (West and Erdos, 1988). These spore 
coat proteins are contained in secretory vesicles (Erdos and 
West, 1989; West and Erdos, 1988), which appear to be intact 
since they are resistant to proteolysis by Proteinase K 
unless they are treated with 0.1% Triton X-100 (West et al., 
1986; Q.H. Yang and CM. West, unpublished data). In other 
studies, N-acetyl glucosaminyltransf erase activity was 
inhibited by EDTA in the P100 fraction only when assayed in 
the presence of detergent, suggesting that Golgi-like 
vesicles in the P100 were closed (R.B. Mandell and CM. 
West, unpublished observations) 

FP21 is Unrelated to Other Known Cytoplasmic Proteins 

The possible relationship of FP21 with other known 
Dictyostelium proteins was investigated. I examined the 
reactivity of antisera raised against discoidin I and II 
(Erdos and Whitaker, 1983) and against gp24 (Knecht et al., 
1987) for FP21. On Western blots, antiserum against gp24 



68 
recognized a band that migrated with a slower mobility than 
metabolically labelled FP21 (not shown). The possibility of 
FP21 being the lectin discoidin was examined, since it is 
primarily present in the cytosol and exhibits weak affinity 
for L-fucose (Erdos and Whitaker, 1983; Bartles and Frazier, 
1980). Metabolically labelled FP21 from HL250 was 
electrophoresed in a 15% SDS polyacrylamide gel, while a 
replicate lane was blotted onto nitrocellulose paper and 
immunoprobed using an anti-discoidin antiserum. The 
antiserum recognized a band of higher MW than FP21 with 
mobility slower than trypsin, reproducing results reported 
by others where discoidin I was shown to have a slower 
mobility than trypsin in 15% SDS polyacrylamide gels 
(Kohnken and Berger, 1987). Migration of purified discoidin 
in SDS-PAGE differed upon boiling of the sample, migrating 
as a tetramer (ca. 100 kD) when samples were not boiled, 
whereas metabolically labelled FP21 was found to migrate as 
a discrete peak of radioactivity ahead of trypsin regardless 
of boiling (Q.H. Yang and CM. West, unpublished data). 
FP21 could be partially purified by HPLC DEAE 
chromatography. Metabolically labelled S100 was 
fractionated on an anion exchange column and FP21 recovery 
monitored by counting of SDS-PAGE slices. FP21 eluted at 
0.5 M NHAc with an increase of 24-fold the specific 

4 

activity relative to the starting sample (data not shown) . 
Discoidin eluted earlier than FP21 from the HPLC DEAE column 



69 
and no radioactivity was found associated with discoidin, 
suggesting it does not bind to discoidin during 
purification. I conclude that FP21 is not related to 
discoidin or any discoidin isoforms, or gp24, and does not 
bind to any discoidin isoforms. 

Oligosaccharide Studies 

FP21 from Ax3 and HL250 yield similar size 
glycopeptides after pronase digestion . Pronase digested 

gel-purified FP21 that had been metabolically labelled from 

Ax3 and HL250 were compared (figure 3-4). More than 50% of 

the radioactivity from both sources eluted as a major peak 

with a relative elution coefficient (Rev) of 0.42 and 0.43, 

respectively (ca. 5.5 glucose units). In the case of FP21 

derived from Ax3, the rest of the radioactivity eluted 

earlier in the void volume and distributed into minor peaks. 

The digestion products from HL250 FP21 yielded a major peak, 

with the rest of the radioactivity eluting earlier, and less 

than 10% eluting after the major peak, although some 

variability was observed in different runs regarding the 

minor peaks. Radioactivity eluting at an earlier position 

than the major peak may be explained by incomplete 

digestion. The minor amount of radioactivity that eluted at 

a later position may be due to breakdown of the 

oligosaccharide. These results indicate that the main 

glycopeptides derived from Ax3 and HL250 have the same 



Figure 3-4. Gel filtration chromatography of FP21 glycopeptides . 

Ax3 and HL250 vegetative cells were metabolically labelled 
with 2 jiCi/ml of [ 3 H]fucose. Cells were lysed, fractionated 
into an S100 and P100, and FP21 isolated by SDS-PAGE and 
electroelution from the S100. The samples were exhaustively 
digested with pronase and analyzed by BioGel P-4 gel 
filtration. Data obtained from one representative 
experiment. 

Panel A. Glycopeptides derived from Ax3 FP21, arrow 
identifies major peak with a Rev of 0.42. Vo, 32; Vi, 142. 

Panel B. Glycopeptides derived from HL250 FP21, arrow 
identifies major peak with a Rev of 0.43. Vo, 26; Vi, 166. 



71 



600 




t 1 1 1 r 

60 80 100 120 140 

fraction 



240 




20 40 60 80 100 120 140 160 

fraction 



72 
sizes, and suggest that both strains form in vivo the same 
oligosaccharide when grown in fucose-containing media. 

Oligosaccharide in FP21 is Q-linked . An enzymatic and 
a chemical approach were used to determine whether the 
fucose-containing oligosaccharide in FP21 is N-linked or 0- 
linked. I first tried the enzyme PNGase F. The minimum 
reguirement of PNGase F is a di-N-acetylchitobiose core 
(Chu, 1986; Tarentino et al., 1985). This enzyme has a 
broad specificity and can cleave most asparagine linked N- 
glycans (including high mannose and complex multibranched 
oligosaccharides) provided they are not located at the amino 
or carboxy termini (Chu, 1986). Gel-purified FP21 from 
metabolically labelled Ax3 was digested with PNGase F. As 
shown in figure 3-5, the fucose label eluted in the void 
volume, while control substrates were guantitatively 
digested as determined by SDS-PAGE (not shown). FP21 
trypsinized prior to digestion with PNGase F also eluted in 
the void volume (not shown). The inability of PNGase F to 
release radioactivity from FP21 suggested that either the 
fucose-containing oligosaccharide was not N-linked or the 
oligosaccharide was insensitive to the enzyme. 

I then considered the possibility that the 
oligosaccharide in FP21 was 0-linked. Metabolically 
labelled gel purified FP21 was subjected to mild alkaline, 
reducing conditions, to release intact 0-linked 



Figure 3-5. Gel filtration chromatography of PNGase F digests. 

Ax3 vegetative cells were metabolically labelled with 2 
jiCi/ml of [ 3 H]fucose, an S100 was prepared, and FP21 was 
isolated by SDS-PAGE and electroelution. FP21 digested with 
PNGase F as described in Materials and Methods, and analyzed 
by BioGel P-4 gel filtration. Nine drops fraction were 
collected, instead of the usual 12 drops; [ H]mannose was 
used to determine Vi . 



74 



Q. 
Q 



1200 



900 - 



600 - 



300 - 




100 120 140 160 180 

fraction 



75 
oligosaccharides (fi-elimination) . Under the conditions 
employed, N-linked oligosaccharides are insensitive to 
chemical release (Biermann, 1988). More than 95% of the 
radioactivity was released from Ax3 (Rev 0.48) and HL250 
(Rev 0.49) in vivo labelled FP21 and it was resolved as one 
peak with a size of 4.8 glucose unit (figure 3-6, panels A 
and B) . The oligosaccharide appeared to have been released 
by fi-elimination and thus is concluded to be O-linked. 
Consistent with the pronase digestion studies reported 
above, Ax3 and HL250 yielded a similar size oligosaccharide, 
supporting the idea that both strains produced the same 
oligosaccharide. The slightly smaller size of the 
oligosaccharide compared to the glycopeptide is consistent 
with the notion that the glycopeptide consisted of one or 
more amino acids and the oligosaccharide chain. If the 
glycopeptide resulting from pronase digestion consisted of 
the oligosaccharide attached to two or more amino acids, it 
was possible that mild alkaline hydrolysis under reducing 
conditions resulted in further hydrolysis of the remaining 
polypeptide backbone yielding one amino acid and the 
oligosaccharide. This would yield a smaller radioactive 
species, with concomitant increase in Rev. To investigate 
this possibility, I employed harsher chemical conditions. 
In vivo labelled FP21 from Ax3 was gel purified and 
subjected to strong alkaline hydrolysis in the presence of 
sodium borohydride, which cleaves N- and O-linked 



Figure 3-6. Gel filtration chromatography of FP21 
oligosaccharides . 

Vegetative cells were metabolically labelled with 2 jiCi/ml 
of [ 3 H]fucose, an S100 was prepared, and FP21 was isolated 
by SDS-PAGE and electroelution. Gel purified FP21 from Ax3 
and HL250 were subjected to fi-elimination or strong alkaline 
hydrolysis followed by fractionation by gel filtration 
chromatography. Data obtained from one representative 
experiment . 

Panel A. fi-elimination of Ax3 FP21. Vo, 36; Vi, 141. 

Panel B. ^-elimination of HL250 FP21. Vo, 30; Vi, 140. 

Panel C. Alkaline hydrolysis of Ax3 FP21. Vo, 38; Vi, 134. 



77 



6000 



4000 



Q. 

Q 



2000 - 




o -pznsnjBaa#j3 



nnmnpsi 



20 40 60 80 100 120 140 
fraction 



800 



600 - 



Q 400 



200 - 



B 




fraction 



2500 




20 40 60 80 100 120 140 

fraction 



78 
oligosaccharides (Zinn et al., 1978; Biermann, 1988). The 
results of this reaction are seen in figure 3-6, panel C. 
More than 75% of the radioactivity was released, eluting 
with a Rev of 0.49. Most of the remainder of the 
radioactivity eluted in the void volume. I expected to see 
a change in Rev by strong alkaline hydrolysis compared to 
mild alkaline hydrolysis if mild alkaline hydrolysis did not 
release the oligosaccharide, but hydrolysed the protein. 
The fact that similar results are obtained by mild and 
strong conditions suggested that fi-elimination occurred to 
release the oligosaccharide. Since mild alkaline hydrolysis 
had been shown earlier not to cleave N-linked sugars 
(Biermann, 1988), I conclude that the oligosaccharide in 
FP21 is linked via an O-linkage. The released 
oligosaccharide eluted as an asymmetrical peak, both by mild 
and strong alkaline hydrolysis. These results suggest that 
there is more than one type of oligosaccharide in FP21 that 
differ slightly in size. These results also suggest that 
the slight heterogeneity seen in the glycopeptide size may 
reflect amino acid heterogeneity. 

Comparison of qlycopeptides derived from vesicular and 
cytosolic material . To further address the possibility of 

FP21 arising by contamination of the S100 from the P100 

fraction, glycopeptides derived from the Ax3 P100 were 

compared to gel purified pronase digested FP21 from Ax3. 

P100 derived glycopeptides were obtained in three different 



79 
manners. Metabolically labelled P100 was subjected to SDS- 
PAGE and the material that comigrated with FP21 was gel 
purified, pronase digested, and fractionated by gel 
filtration chromatography. Entire P100 from metabolically 
labelled cells was digested in the presence or absence of 
detergent, and analyzed by BioGel P-4 gel filtration. As 
seen earlier in figure 3-4, panel A, the S100 digest eluted 
mainly as a single peak with a Rev of 0.42; on the other 
hand, the digest from the FP21-comigrating P100 material, 
fractionated as a major peak of 0.50 Rev (figure 3-7, panel 
A) . An analysis of glycopeptides from the entire P100 
digestion showed a different elution profile from the FP21 
digestion (figure 3-7, panel B) . Note than in this 
chromatograph there is a minor peak of 0.51 Rev, consistent 
with the idea that the major peak seen in panel A is a minor 
component of the entire P100 glycopeptide repertoire. Also 
approximately 40% of the radioactivity eluted with the void 
volume, suggesting it may be resistant to pronase digestion. 
Digestion of the entire P100 fraction was carried out in the 
presence of Triton X-100, to determine if solubilization of 
the sample yielded a different digestion profile, by 
facilitating accessibility of the enzyme to the substrates 
(figure 3-7, panel C) . The overall profile is similar, with 
more than 30% of the radioactivity eluting at the void 
volume, and a peak with a Rev of 0.51 is still a minor 
component of the glycopeptides released. West et al. (1986) 



Figure 3-7. Gel filtration chromatography of P100 glycopeptides . 

Vegetative Ax3 cells were metabolically labelled with 2 
/iCi/ml of [ 3 H]fucose. A P100 was prepared and the 21 kD MW 
material that comigrated with FP21 on SDS-PAGE was 
electroeluted and pronase digested. In another assay, 
entire P100 was pronase digested in the absence or presence 
of 0.1% Triton X-100. After pronase digestion, samples were 
subjected to BioGel P-4 fractionation. Arrow identifies the 
position with a Rev of 0.51. 

Panel A. Glycopeptides from 21 kD MW PlOO-derived material. 
Vo, 34; Vi, 162. 

Panel B. Glycopeptides from entire pronase-digested P100. 
Vo, 36; Vi, 142. 

Panel C. Glycopeptides from entire P100 digested with 
pronase in the presence of Triton X-100. Vo, 32; Vi, 146. 



81 



£UUU - 


A. 




1500 - 


I 


E 1 000 - 
Q 

500- 


Vo 
1 


. Vi 

1 * 


0- 







20 40 60 80 100 120 140 160 
fraction 



350 




20 40 60 80 100 120 140 160 

fraction 



Ql 
Q 



2000 - 
1500 - 


Vo 

> 




1000 - 

500- 

- 


-_ V IrvWUV-.- 


Vi 



20 40 60 80 100 120 140 160 

fraction 



82 
have reported the existence of pronase-resistant material in 
the particulate fraction of vegetative cells, so it seems 
possible that the pronase-resistant material eluting in the 
void volume is, or is related to, the smear previously 
described, although SDS-PAGE analysis will be needed to 
confirm this supposition. 

Fucose is Covalently Bound to FP21 

The fact that the radioactivity in FP21 was not 
released by boiling in SDS/ fi-mercaptoethanol, nor after 
boiling in SDS under reducing conditions followed by SDS- 
PAGE, suggested that 3 H was covalently bound to in vivo 
labelled FP21. Nevertheless, there have been reports of 
covalent-bound enzymatic intermediates in the literature 
(Scrimgeour, 1977). Although ES (enzyme-substrate) 
intermediates are very reactive and usually cannot be 
isolated without some sort of stabilization or chemical 
trapping technigue, the possibility that FP21 is really a 
cytosolic fucosyltransferase that binds GDP-fucose or other 
fucose metabolites covalently was considered. The Rev of 
the radioactivity released by mild alkaline hydrolysis 
suggests that it is not related to GDP-fucose or fucose 
because it elutes with a different Rev than GDP-fucose or 
fucose. Additional evidence that 3 H is present as fucose 
was presented in Chapter II, where it was shown that more 



83 
than 95% of the macromolecular-associated radioactivity from 
metabolically labelled cells migrated as authentic fucose. 

FP21 is Present in Migrating Slug Stage Cells 

To investigate whether FP21 was present also in 
developing cells, HL250 amoebae were plated for development 
on nuclepore filters and 9 hours after plating the filters 
were lifted and cells were metabolically labelled by 
placement on 100 jiCi of [ 3 H] fucose. After 5 hours of 
exposure to [ 3 H] fucose, filters were lifted again, carefully 
washed from 3 H label, and cells were allowed to continue 
development for two more hours. Cells were then harvested, 
disaggregated, and fractionated into an S100 and P100 
fractions, run in an SDS-PAGE, and the gel cut and the 
pieces counted. As shown in figure 3-8, a fucosylated 
macromolecule is preferentially fucosylated in the cytosol 
and it has the same mobility in SDS-PAGE as FP21. Thus, it 
seems that FP21 fucosylation is not restricted to the growth 
phase of Dictyostelium. 

Discussion 
In this chapter I have presented evidence for the 
existence of a fucosylated single molecular weight species, 
which has been termed FP21 based on its mobility as 
determined by SDS-PAGE. This protein cofractionates with 



Figure 3-8. Incorporation of [ 3 H]fucose into macromolecular 
species of slug stage cells. 

HL250 cells were harvested, plated on filters for 
development, and exposed to [ H]fucose for 5 has described 
in the text. Cells were then collected, disaggregated, 
filter-lysed, and S100 and P100 fractions were prepared and 
analyzed by 7-20% linear gradient SDS-PAGE. 70 uq and 24.6 
uq of protein were electrophoresed for the S100 and P100, 
respectively. Open circles, P100; closed circles, S100; 
arrow, migration position of trypsin. 



85 



1600 




6000 



4000 _. 



0. 

Q 



o 
o 



- 2000 a. 



gel slice 



86 
the high speed supernatant, S100, being the major 
fucosylated species in this fraction. The recoverability of 
FP21 after TCA precipitation, HPLC anion exchange 
chromatography, boiling in SDS/fi-mercaptoethanol, and 
methanol/acetic acid fixation of the gels suggested a 
covalent nature for the association of radioactivity. It is 
unlikely that FP21 is a cytosolic fucosyltransf erase that 
binds GDP-[ 3 H]fucose or another fucose metabolite 
covalently, since FP21 does not copurify with the cytosolic 
fucosyltransferase (CM. West, unpublished results). 
Employing enzymatic and chemical analysis, the 
oligosaccharide in FP21 was examined and characterized as a 
small (4.8 glucose unit) oligosaccharide. The 
oligosaccharide appeared to be O-linked based on its 
insensitivity to PNGase F and the releasability from FP21 
under alkaline, reducing conditions. Ax3 and HL250 produced 
FP21-derived glycopeptides and oligosaccharides of similar 
size, suggesting both strains carry out similar 
modifications in vivo, despite the starvation for fucose in 
HL250. Thus, it appears there are no competing reactions 
for the unfucosylated oligosaccharide, unlike the case for 
outer fucose in N-linked glycans from mammals (Paulson et 
al., 1978). Based on antiserum specificity, molecular 
weight, and/or fractionation by HPLC anion exchange 
chromatography, FP21 was shown to be a protein unrelated to 
discoidin or gp24. Finally, a 21 kD fucoprotein was present 



87 
in the cytosol of developing cells, suggesting FP21 was not 
limited to the vegetative stage in Dictyostelium, and was 
fucosylated during development. 

I believe that FP21 is recovered in the S100 because it 
resides in the cytosol in living cells, and not as default 
location from ruptured vesicles, for several reasons. The 
S100 fraction was shown to be equivalent to the cytosol and 
essentially devoid of organellar markers. S100 and P100 
fractions from [ 3 H]fucose metabolically labelled cells 
exhibited a different radioactive profile by SDS-PAGE. 
Sonication of the P100 fraction failed to release FP21 into 
the supernatant. FP21 appeared to be endogenous to the 
cytosol and not derived from organellar vesicles, because 
control experiments suggested there was no generalized 
breakage of vesicles during the preparation of the cytosolic 
fraction. In addition, as a control for contamination from 
P100 material, glycopeptides derived from comigrating 21 kD 
MW species from the P100 fraction were compared with FP21 
glycopeptides. The fucopeptide in FP21 (ca. 5.5 glucose 
unit) does not seem to be a product of vesicular 
fucosylation, since it is not shared by macromolecules of 21 
kD MW in the P100 fraction which yielded a major peak with a 
different Rev (ca. 4.3 glucose unit) than the one derived 
from FP21. When the entire P100 fraction was subjected to 
Pronase digestion, a heterogeneous mixture of fucosylated 



88 
species (in accordance with Tsurchin, et.al. 1989) was 
obtained with sizes unlike that of FP21 glycopeptides. 

The designation of compartmentalization of a protein as 
cytosolic is difficult since the cytosol is the site of 
localization after disruption of organellar vesicles. This 
task is complicated in the case of glycoproteins and 
glycosylation enzymes, which are usually described as 
components of the secretory pathway. However, some 
glycoproteins have been identified as cytosolic, and 
generally accepted as such (Hart et al., 1989a; Hart et al., 
1989b) . My studies report the existence of a fucosylated 
cytosolic protein. However, these results do not exclude 
the possibility of FP21 being present in other locations 
topologically continuous with the cytosol, such as the 
nucleus, or being synthesized elsewhere and transported. 
Due to its small size, FP21 could, in theory, be able to 
diffuse freely into the nucleus. 

Since glycoprotein fucosylation has been shown to take 
place in enclosed organelles of the secretory pathway 
(Hirschberg and Snider, 1987), the identification of FP21 in 
the cytosol raises the question of where in the cell is 
fucosylation of FP21 taking place. One scenario would have 
FP21 being fucosylated in organellar vesicles (presumably 
the Golgi apparatus) and subsequently transported to the 
cytosol, while another would postulate the presence of a 
fucosyltransf erase that localized in the cytosol with FP21. 



89 
I consider these possible alternatives in the following 
chapter and present evidence for the presence of a 
fucosyltransf erase in the cytosol responsible for FP21 
f ucosylation . 



CHAPTER IV 
EVIDENCE FOR A CYTOSOLIC FUCOSYLTRANSFERASE 



Introduction 

In the preceding chapter, I identified a fucosylated 
protein in the cytosol, FP21. The presence of FP21 in the 
cytosol challenges the prevailing belief that fucoproteins 
are restricted to the cell surface and lumenal compartments 
of the cell. Even though in the past three decades evidence 
has been accumulating on the presence of glycoproteins and 
fucoproteins in non-lumenal locations (Hart et al., 1989a; 
Hart et al., 1989b), to my knowledge, no one has shown the 
existence of a fucosyl transferase in the cytosol. One 
possibility is that fucosylation is restricted to the 
microsomes, and cytosolic fucoproteins are 

posttranslationally transported back across the membrane to 
the cytosol. 

However, even though there is no previous evidence for 
cytosolic fucosylation there is precedent for glycosylation 
in the cytosol. Studies on the biosynthesis of nuclear pore 
proteins bearing O-GlcNAc suggested that the sugar was added 
to the proteins within 5 min of their synthesis and before 
they became associated with membranes (Davis and Blobel, 
1987). These data suggested that the activity responsible 

90 



91 
for the addition of O-GlcNAc was in the same topological 
compartment where translation takes place, the cytosol. 
Thus it is possible that fucosylation may take place in the 
cytosol, but it has escaped detection by previous 
investigators for a variety of reasons. One of the 
difficulties in assaying cytosolic enzymes is that the 
endogenous acceptors for the enzymes may be present in low 
quantities in the cell, complicating purification of large 
amounts for use as substrates. If acceptors are already 
fucosylated, in vitro assays that utilize endogenous 
acceptors would not detect enzymatic activity. Hart and 
coworkers have circumvented this problem with the use of 
synthetic peptides with a sequence based on O-GlcNAc 
glycosylation sites (Hart et al., 1989b). They have 
identified an enzymatic activity capable of O-linked GlcNAc 
transfer in rat hepatocytes that was recovered in both the 
soluble and membrane fractions (Haltiwanger et al., 1990). 
The membrane-associated activity was releasable by high salt 
treatment and oriented towards the cytosol, not the lumen of 
the vesicles (Haltiwanger et al., 1990). 

With the help of the conditional fucosylation mutant 
HL250, I have addressed the existence of a fucosylation 
pathway in the cytosol. Total protein in HL250 is 
underfucosylated relative to the normal strain, so it was 
reasoned that it would be a useful strain to assay 
fucosylation in vitro due to the availability of 



92 
macromolecular acceptors. In this chapter I present 
evidence for a fucosyltransferase that partitions with FP21 
in the cytosol. The fucosyltransferase was distinguished 
from vesicular fucosyltransferase activity by several 
criteria, and was characterized using hydrophobic synthetic 
analogs. 

Materials and Methods 
Materials 

GDP-[U- 3 H]fucose (6.6 Ci/mmol) and GDP-[U- 14 C]fucose 
(250 mCi/mmol) were from New England Nuclear (more than 90% 
of the radiolabel was in the form of the R anomer, as 
indicated by the manufacturer). Reagent grade KC1, MnCl 2 , 
CaCl , BaCl 2 and MgCl 2 were from Mallinckrodt; GDP-fi-fucose 
from Biocarb (stored frozen as a concentrated stock); GDP-cr- 
fucose (stored frozen as a concentrated stock), Tween-20, 
CoCl 2 , Dowex-2 (2x8, minus 400, chloride form), Triton X- 
100, chymostatin, pepstatin, NBZ-phenylalanine, bovine serum 
albumin (BSA), and all p-nitro-phenyl acceptors were from 
Sigma; Na 2 EDTA, FeCl 3 , formic acid, and trichloroacetic acid 
were from Fisher. Cations and Na 2 EDTA were stored as 
concentrated 500 mM solutions at 4°. Hydrophobic synthetic 
acceptors were generously provided by Monica Palcic. Making 
and regeneration of Dowex-2 formate form column was as 
described for Dowex-1 in Materials and Methods, Chapter II. 
The concentrations of Triton X-100 and NP-40 are expressed 



93 
as volume/volume (v/v), all others are expressed as 
weight/volume (w/v) unless specified otherwise. 

Strains and Conditions of Growth 

Dictyostelium discoideum strains Ax3 (from S. Free) and 
HL250 (from W.F. Loomis) were grown on HL-5 # a complete 
medium that contains glucose, yeast extract, and proteose 
peptone (Loomis, 1971). Ax3 is the normal strain and HL250 
is a mutant obtained from Ax3 by N-methyl-N' -nitro-N- 
nitrosoguanidine mutagenesis (Loomis, 1987). HL250 lacks 
the enzyme activity that converts GDP-mannose into GDP- 
fucose which results in a lack of cell fucose (Gonzalez- 
Yanes et al., 1989; Chapter II). In all experiments cells 
were collected at the logarithmic growth phase, with a cell 
density of 1 to 9 x 10 6 cells/ml. 

Cell Lysis and Fractionation 

Vegetative and slug stage cells were fractionated into 
S100 and P100 fractions as described in Materials and 
Methods, Chapter III. 

Fucosyltransferase Assay 

Fucosyltransferase activity was assayed immediately 
after obtaining the S100 and P100 fractions. Fractions were 
found to be sensitive to freezing and thawing, and up to 70% 
of the fucosyltransferase activity could be lost. The 



94 
standard fucosyltransferase assay contains 30 /il of extract 
(100-350 /ig of protein), 0.35 nM GDP-fi-[ 14 C]-fucose, 5 mM 
MgCl 2 , 0.25 mM NaF, and 5 mM ATP in 50 mM MES (titrated with 
NaOH to a pH of 7.4) in a 50 )il volume incubated at 30° for 
the specified amount of time. Endogenous macromolecules 
were used as acceptors. To terminate the assay, 1 ml of 
ice-cold 15% TCA was added to each sample along with 50 vq 
BSA (to serve as carrier protein) and the precipitate 
collected on 2.4 cm GF/C glass filters by vacuum filtration, 
washed with 10 ml 10% ice-cold TCA, 10 ml acetone, and 
counted after air-drying inside the vials for approximately 
30 min using 10 ml of Bio-HP LC scintillation fluid 
(Fisher). Background was subtracted from experimental 
values, and was determined as the amount of TCA-precipitable 
radioactivity at time zero; it was usually between 20-40 
dpm. When indicated, the disodium EDTA salt was used. In 
preliminary trials, 5 mM Mg ++ was found to support maximal 
activity, and this concentration was used in all the assays 
unless indicated. GDP-fucose had been shown previously to 
be only slightly decomposed under similar conditions (Nunez 
and Barker, 1976). Nevertheless, the extent of GDP- 
[ 14 C]fucose hydrolysis during the fucosyltransferase assay 
was examined by carrying out the reaction for two hours and 
separating products on a Dowex-2 formate column by 
sequentially eluting with 5 ml of water, 3 M formic acid and 
15 M formic acid as described (Sommers and Hirschberg, 



95 
1982). After 2 hours of incubation less than 20% of the 
initial GDP-[ 14 C]fucose had been hydrolyzed. Incorporation 
of 14 C did not exceed 30% of the initial radioactivity on 
any given experiment. For analysis by gel electrophoresis 
of the endogenous acceptors of the in vitro 

fucosyltransferase activity, the reaction was terminated by 
3 min boiling in sample buffer. For the determination of pH 
optima experiments, assays were buffered using concentrated 
solutions of MES previously adjusted to different pH values 
with either HC1 or NaOH. The final pH value of each 
reaction was determined on an equivalent reaction mixture 
lOOx the volume, without GDP-[ 14 C]fucose. Where indicated, 
the S100 was desalted on a 0.85 x 13 cm on a BioRad BioGel 
P-2 column (200-400 mesh) equilibrated with 50 mM MES, pH 
7.4, titrated with NaOH, at 4° with a flow rate of 0.5 
ml/min. If supplied, synthetic acceptors and/or FP21 were 
previously dried down onto the bottom of the assay tubes in 
a vacuum centrifuge. Acceptors were subsequently 
resuspended in water or in the reaction mixture. Results 
are expressed as average of two determinations (variations 
in the duplicates did not exceed 15% of the average value) 
or average of three measurements ± standard error of the 
mean (s.e.m.). Calculations of K and V were done by the 

* * m max •* 

Hanes single reciprocal plot ([S]/v vs. [S]) as discussed by 
Henderson (1985) . 



96 
Incorporation into hydrophobic synthetic acceptors was 
determined as described by Palcic et al. (1988). The assay 
was carried out as for endogenous acceptors, but GDP- 
[ 3 H]fucose was used instead of GDP-[ 14 C] fucose and the 
reaction was terminated by the addition of 1 ml ice-cold 
water. The reaction mixture was loaded onto a C 18 SepPak 
column (Waters) under vacuum, and eluted with 6 successive 5 
ml aliquots of water, and four 5 ml aliquots of methanol. 
Eluates were counted by addition of 15 ml of ScintiVerse LC 
(Fisher) . In initial trials I determined that the 
radioactivity eluted in the first methanol fraction, so in 
subsequent experiments only the first methanol fraction was 
used for determination of radioactivity incorporated. All 
extracts were assayed in the absence of exogenous acceptor 
and this value (usually about 20% of the dpm incorporated) 
subtracted from experimental value to determine substrate- 
dependent incorporation. 

Purification of FP21 

FP21 was purified in the following manner for 
preparations which were to be added back to cytosolic 
fractions to measure fucosylation acceptor activity. 
Starting with 8 x 10 10 cells, an S100 cytosolic fraction was 
prepared from the mutant HL250. An aliquot of the fraction 
(approximately 0.7% of the total volume) was incubated with 
GDP- [ 14 C] fucose and allowed to fucosylate FP21 with 



97 
[ 14 C]fucose. Incorporation into FP21 was confirmed by 
electrophoresing an aliquot and counting of SDS-PAGE slices. 
The radiolabelled aliquot was mixed with the rest of the 
unlabelled preparation and subjected to (NH 4 ) 2 S0 4 
fractionation. The 70-80% cut was dissolved in and dialyzed 
against 100 mM NH 4 Ac, applied to a 14 ml bed of the strong 
anion exchanger A25-QAE-Sephadex / and eluted with an 
ascending gradient up to 1.5 M NH 4 Ac. [ 14 C]FP21 eluted at 
input buffer concentration of 0.49 M. This preparation was 
then concentrated and desalted on Centricon and/or 
Centriprep cartridges with nominal 10 kD MW cutoffs, and 
then applied to an HPLC gel filtration column (8 x 300 mm 
Toya Soda TSK GW-300) equilibrated in 100 mM NH 4 Ac, with a 
flow rate of 0.5 ml/min. Sample was clarified by 
centrifugation at 10k x g for 10 min prior to injection. 
Radioactivity from the concentrated QAE-Sephadex eluate 
eluted between the 14 kD and 29 kD MW standards. Fractions 
were analyzed by SDS-PAGE using 15% polyacrylamide gels and 
counting of the gel slices. For addition of purified FP21 
to cell extracts, HPLC gel filtration fractions were brought 
to dryness in a vacuum centrifuge, redissolved in dH 2 0, and 
brought to dryness again, in the 1.5 ml microcentrifuge tube 
that was going to be used for the assay. 



98 
fi-elimination of In Vitro Labelled Acceptor 

S100 extracts from HL250 were fucosylated in vitro. To 
corroborate that I obtained 21 kD MW fucosylated product 
from the in vitro reaction, l/25th of the reaction was 
terminated by 3 min boiling in sample buffer and analyzed by 
SDS-PAGE. The remainder of the sample (containing 
approximately 10* dpm) was stored at -80° until ready to 
use. The reaction mixture was centrifuged for approximately 
2 h in a centricon filter to reduce unused GDP-[ 14 C]fucose. 
After concentrating the volume to 200 /il, li-elimination was 
carried out as described in Materials and Methods, Chapter 
III. 

PNGase F Digestion of In Vitro Labelled FP21 

FP21 was fucosylated in vitro as described above for R- 
elimination. After the volume was concentrated, PNGase F 
digestion was carried out as described in Materials and 
Methods, Chapter III. 

Results 
Cytosolic Fucosyltransferase Activity 

The presence of FP21 in the cytosol suggested that a 
fucosyltransferase might also be located there. To 
investigate this possibility, HL250 cells were fractionated 
to yield cytosolic supernatant (S100) and organelle (P100) 
fractions. The fractions were analyzed for their ability to 



99 
transfer [ 14 C] from GDP-[ 14 C]fucose into TCA precipi table 
endogenous material. As shown in figure 4-1, the cytosolic 
activity was dependent on time and protein content. The 
cytosolic fucosyltransferase activity had the properties of 
being enzyme-mediated. Table 4-1 shows the effect of 
boiling, denaturants and temperature on the cytosolic 
fucosyltransferase activity. While the non-ionic detergent 
Triton X-100 inhibited all activity, Tween-20 was only 
slightly inhibitory. 30° was the optimal temperature of 
those tested (22°, 30°, and 37°). Consistent with an 
enzyme-mediated process, only unlabelled GDP-fl-fucose was 
able to inhibit incorporation of radioactivity proportionate 
to its relative concentration (GDP-a-fucose was without 
effect), demonstrating the stereospecif icity of the enzyme 
(lower section of table 4-1). It also implies a 
fucosyltransferase that catalyses an alpha-fucosyl linkage 
is being assayed. 

Earlier I explored the possibility of FP21 arising by 
contamination from the vesicular fraction. The same 
question was asked about the fucosyltransferase activity in 
the cytosol, since known fucosyltransf erases are Golgi 
enzymes and activity was detectable in the P100 (see next 
section) . Hence, I tried to deplete the S100 of 
fucosyltransferase activity by centrifuging at 170k x g for 
2.5 hours (instead of 1 hr at 100k x g) . This step was used 
to sediment any population of small or low density vesicles 



Figure 4-1. Fucosylation of endogenous acceptors by S100 
fraction. 

Vegetative HL250 cells were harvested, homogenized, and an 
S100 obtained as described in detail in Materials and 
Methods. The indicated amount of S100 protein was incubated 
in the presence of 0.36 /jM GDP-[ 14 C] fucose, 5 mM MgCl 2 , 5 mM 
ATP, 0.25 mM NaF, in 50 mM MES, pH 7 . 4 for the indicated 
amount of time. Fucose incorporation was calculated from 
the amount of TCA-precipitable [ 14 C] radioactivity. Results 
expressed as the mean of three determinations + s.e.m. 

Panel A. Effect of time on fucosylation; 159 uq protein. 

Panel B. Effect of protein concentration on fucosylation; 
30 min assay. 



101 



3 
o 

Q. 
O) 

E 

CO 

o 
o 



o 

E 

Q. 




time (min) 




100 150 200 250 300 350 



M.g protein 



102 



P 



P 
u 

03 
d> 

to 

03 
M 

d) 
4H 

to 

c 

03 
M 

P 

H 

in 
O 
u 

3 



U 
•H 

rH 
O 

to 
o 

P 
>i 

u 

d> 
£ 
+J 

c 
o 

to 

p 

c 

1 

p 

03 
d> 
M 

P 

*J 

C 
(1) 

CD 



4-1 

o 

U 

d> 

«w 

4-1 
W 



I 



XI 
03 

Eh 



+J 

•H 
> 
•H 
4J 

U 

03 

CD 
> 

•H 
+J 

03 
H 
CD 

M 



C 

•H 
S 

o 
n 

tr 

e 



Q) 

to 

O 

u 

3 



03 

c 
0) 
E 

•H 
5-1 
0) 

a 
x 
ai 



o 

e 



2 

=1 



d) 
to 
o 

CJ 

3 
4-1 

u 



I 

Oi 

8 



c 
o 



•a 
c 
o 
u 



ioHH«nion«) cn in 
oooovoor~«* oo 



oooooooo 

V 



VO CN CN VD \D 

n rn n n n 



OrHi-HOOVOVOVO 



c 
o 

•rH 

P 
03 

U 



o 
o 

rH 
I 

X 

c 

o 
p 



u 

Eh 



Eh 

a 

2nm# 



O 

c 

03 
■C 
P 
0) 

<#> o o 
o cn r» 



.-H O 



MOHtNinoooH men 

OS O O CN • O • • • O 

• • • • O • CN VO r-4 • 

OOOOi-HmCNiH CN rH 



nnroinvo^tCNro oooo 

* CN CN • • • • • • • 

r-» . •co«o«*iHin oo 

HmoidHinnn cncn 






o o 



d) 
to 
o 
u 

3 



4H 4H 

I I 

CQ 

1 I 

a, a, 

Q Q 

O O 

as s 

a a. 



_f*G 



°ZB° 



to cn 



u 



d) 



CinjjooHNn *£>vo 



103 
that were not sedimented before (if existent) and that could 
have contained fucosyltransferase activity. To preserve the 
intactness of the P100 vesicles, additional protease 
inhibitors (Goodloe-Holland and Luna, 1987) from those 
routinely used, were utilized during cell fractionation. 
After these measures activity was still recovered in the 
cytosol at similar levels (table 4-2). Additional evidence 
supporting the notion that the vesicles in the P100 are not 
damaged during filter lysis and centrifugation is presented 
in Chapter III in the section of the origin of FP21. Taken 
together, these results suggest that vesicles were not 
measurably damaged during the isolation procedure; 
therefore, the fucosyltransferase activity is probably 
endogenous to the cytosol . 

Comparison Between the P100 and S100 Fucosyltransferase 
Activities 

In order to compare the S100 and P100 activities, I 

examined the identity of endogenous acceptors and the 

effects of divalent cations, pH, and varying GDP-fucose 

concentration on both fucosyltransferase activities. The 

criteria of differential behavior has previously been used 

to differentiate glycosyl transferases, since it is assumed 

that under similar conditions, enzymes should behave in a 

similar fashion (Campbell and Stanley, 1984; Galland et al., 

1988). Activities were measured in the presence of 

detergent to circumvent any potential problem in substrate 



104 



Table 4-2. Failure to sediment S100 fucosyltransf erase 
activity. 

S100 P100 

100k x g, lh 943 444 

170k x g, 2.5h 1131 484 



Cells were lysed, centrifuged, and fractionated into S100 and 
P100. In this experiment lysis buffer (described in Materials 
and Methods, pH 8.0) was supplemented with 1 mM chymostatin, 
5 jjg/ml peps tat in, and 2 mM NBZ -phenylalanine. Fractions were 
assayed immediately for [ 14 C] incorporation from GDP- [ C]fucose 
and expressed as total dpm incorporated in 30 min into TCA 
insoluble endogenous acceptors. Reaction mixtures contained 
0.36 ^M GDP-fucose, 5 mM MgCl 2 , 240 uq protein, and were 
incubated for 30 min. Results are expressed as average of two 
determinations . 



105 
or cation accessibility. Tween-20 at a concentration of 
0.1% was chosen because it did not inhibit considerably the 
activity in the S100 (table 4-1). 

I first examined the profile of in vitro endogenous 
acceptors by SDS-PAGE analysis. Standard S100 and P100 
fractions from HL250 cells were isolated and added to 
fucosyltransf erase reaction mixtures. After 90 min of 
incubation, reactions were boiled in sample electrophoresis 
buffer, subjected to SDS-PAGE, and the gels cut and counted. 
The profile of radiolabel incorporation in vitro by the S100 
was similar to that observed in metabolic labelling 
experiments, with more than 70% of the radioactivity 
migrating as one discrete peak with a MW of 21 kD (compare 
figure 4-2, panel A with figure 3-1). Incorporation into 21 
kD MW material by the S100 fraction varied from 70-95% in 
different experiments, with the remainder of the 
radioactivity migrating near the dye front or top of the 
gel. In the P100 fraction, radioactivity distributed in two 
peaks, similar to what was seen in metabolically labelled 
P100. To determine if in vitro fucosylated protein had the 
same apparent MW as metabolically labelled FP21, Ax3 gel- 
purified FP21 was mixed with in vitro fucosylated HL250 
S100, as described in the figure legend (figure 4-2, panel 
B) . The migration of in vitro fucosylated material 
coincided with that of metabolically labelled FP21. I 



Figure 4-2. SDS-PAGE profile of endogenous acceptors fucosylated 
in vitro. 

Panel A. SDS-PAGE profile of S100 and P100 endogenous 
acceptors fucosylated in vitro. HL250 cells in logarithmic 
growth phase were harvested, homogenized, and an S100 and 
P100 prepared. Both fractions were fucosylated in vitro in 
the presence of 4.4 j;M and 8.8 /jM of GDP-[ C]fucose for the 
S100 and the PI 00, respectively, 5 mM MgCl , for 90 min. 
Fucosyltransferase reactions were stopped by boiling in 
SDS/fi-mercaptoethanol sample buffer and resolved by 7-20% 
linear gradient SDS-PAGE; the gel was sliced into 2.2 mm 
pieces and counted. Electrophoresis was from left to right. 
138 ^g and 150 vq of protein were electrophoresed for the 
S100 and P100, respectively. Open circles, P100 endogenous 
acceptor species; closed circles, S100 endogenous acceptor 
species; arrow, migration of trypsin. 

Panel B. Comigration of in vitro fucosylated FP21 with 
metabolically labelled FP21 on SDS-PAGE. Ax3 vegetative 
cells grown in [ 3 H]fucose were harvested, lysed, and 
fractionated into an S100 and P100. The S100 was subjected 
to SDS-PAGE and Ax3 FP21 was gel purified and electroeluted. 
Independently, HL250 amoebae were harvested, homogenized, 
fractionated, and the S100 obtained from the fractionation 
fucosylated in vitro in the presence of 0.36 ^/M GDP- 
[ 14 C]fucose, 5 mM MgCl, 140 yq protein, for 30 min. 
Reaction was stopped by mixing with gel purified Ax3 
[ 3 H]FP21 followed by boiling in SDS/fl-mercaptoethanol. 
Samples were coelectrophoresed in a 15% SDS-polyacrylamide 
gel, and the gel cut into 0.5 cm slices and counted. Open 
circles, Ax3 [ 3 H] metabolically labelled gel purified FP21; 
closed circles, [ 14 C] in vitro labelled mutant S100 extract. 



107 



400 




10 



20 30 

gel slice 



2400 



-1800 



-1200 



-600 



o 
o 

CO 



0. 

Q 



40 



1200 




£ 600- 



6 8 10 12 14 16 

gel slice 



108 
interpret these results as an indication that the activity 
that fucosylates FP21 in vivo is being assayed in vitro. 

FP21 was fucosylated in vitro by incubating HL250 S100 
fractions in the presence of GDP-[ 14 C]fucose, and the 
oligosaccharide fucosylation in vitro was examined as before 
for in vivo fucosylated FP21. In vitro fucosylated FP21 was 
digested with PNGase F (not shown) or subjected to mild 
alkaline hydrolysis and analyzed by gel filtration (figure 
4-3, panel A). As was the case with metabolically labelled 
FP21, PNGase F failed to release radioactivity and in vitro 
labelled FP21 digested with PNGase F eluted in the void 
volume. The digestion of the control substrates, fetuin and 
ribonuclease B, was confirmed by SDS-PAGE. On the other 
hand, approximately 20% of the radioactivity eluted with a 
Rev of 0.50, the elution position of the metabolically 
fucosylated oligosaccharide produced by Ax3 (reproduced for 
comparison in figure 4-3, panel B, from figure 3-6, panel 
A) . The remainder of the radioactivity fractionated as 
material of larger size. These results suggested that the 
in vitro fucosylated oligosaccharide in FP21 was also 0- 
linked. The reasons for the discrepancies in size between 
in vivo and in vitro fucosylated oligosaccharides are not 
known, but may be due to incomplete release of the 
oligosaccharide, accompanied by partial hydrolysis of the 
polypeptide. Alternatively, it may indicate the presence of 
oligosaccharides of various sizes. 



Figure 4-3. BioGel P-4 gel filtration chromatography of in vitro 
labelled FP21 oligosaccharide. 

HL250 S100 extracts were incubated in vitro in the presence 
of GDP-[ 14 C]fucose, desalted, subjected to fl-elimination (as 
described in Materials and Methods), and analyzed by gel 
filtration. For comparison, the profile resulting from fl- 
elimination of in vivo labelled Ax3 gel purified FP21 is 
presented in panel B (was panel A in figure 3-6, Chapter 
III) . 

Panel A. fl-elimination of in vitro fucosylated FP21; arrow 
identifies peak with Rev of 0.50. Vo, 38; Vi, 136. 

Panel B. fi-elimination of Ax3 FP21. Vo, 36; Vi, 141. 



110 



2000 



1500 - 



£ 1000- 



500 




20 40 



t — ■ — r 

60 80 100 120 140 

fraction 



a. 
a 



6000 



4000 - 



2000- 




Ill 

To compare the S100 and P100 fucosyltransferase 
activities, HL250 vegetative cells were harvested, 
homogenized, and fractionated into an S100 and P100. 
Fractions were assayed for fucosyltransferase activity under 
different conditions in the presence of Tween-20 (table 4- 
3). The fractions differed in that the S100 
fucosyltransferase activity was approximately threefold more 
efficient (on a per protein basis) than the P100 under 
standard conditions (which contained 0.36 /jM GDP-fucose and 
5 mM MgCl 2 , see Materials and Methods). A major difference 
between the bulk activities was their sensitivity to the 
presence of divalent cations. In the absence of any added 
cation, the P100 retains more than one fourth the activity 
exhibited in the presence of Mg ++ , while the activity in the 
S100 was almost negligible. The presence of EDTA does not 
inhibit further the activity in the P100. One 
interpretation is that the fucosyltransferase activity in 
the S100 is dependent on added Mg ++ , while the activity in 
the P100 is present in the absence and presence of Mg ++ , 
being stimulated by the cation. An alternative explanation, 
is that there are multiple enzymes in the P100, which differ 
in their reguirements for divalent cations. 

Sensitivity to pH is a feature exhibited by enzymes, 
including fucosyltransf erases (Foster, et al. 1991; Kumazaki 
and Yoshida, 1984). HL250 vegetative cells were harvested, 
lysed, and fractionated into S100 and P100 fractions. Both 



112 



Table 4-3. Comparison between the S100 and P100 
fucosyltransf erase activities. 

condition pmol fucose/mq protein/45 min 

S100 P100 

MgCl " 6.95 + 2.60 1.71 + 0.45 

no cation 0.18+0.08 0.68+0.10 

EDTA 0.05+0.03 0.64+0.10 



HL250 amoebae were harvested, filter-lysed, and centrifuged to 
prepare S100 and P100 fractions. Fucosyltransf erase reaction 
mixtures contained 0.36 jiM GDP-[ 14 C] fucose, 0.1% Tween-20, and 
135 uq or 138 uq of protein from the S100 and P100, 
respectively, in the absence of added divalent cations. MgCl 2 
and EDTA were present at 5 mM. *this is the standard assay, 
as described in Materials and Methods. Results are the 
average of three measurements + s.e.m. 



113 
fractions were assayed for fucosyltransferase activity at 
various pH values as described in Materials and Methods. 
The pH profiles of the S100 and P100 fucosyltransferase 
activities in the presence of Tween-20 are shown in figure 
4-4. Activity was maximal for the S100 from pH 6.8 to 7.8 
and for the P100 from pH 6.4 to 7.8. At pH 9.6 the activity 
in the S100 was inhibited more than 20-fold compared to 
maximum (p<0.05), whereas the activity in the P100 was only 
inhibited threefold (p<0.05). The pH-dependent activity 
profiles were not affected by the exclusion of Tween-20 (not 
shown). Thus, while the general profile is similar for the 
activities from the S100 and P100, the cytosolic activity 
was more sensitive to alkaline pH than the P100 activity. 

The dependence of S100 and P100 fucosyltransferase 
activities on the GDP-fucose concentration in the presence 
of Tween-20 was also studied. S100 and P100 fractions were 
prepared from HL250 vegetative cells and assayed for 
fucosyltransferase activity under standard conditions, at 
increasing concentrations of GDP-fucose in the presence of 
0.1% Tween 20 (figure 4-5). The apparent K m for GDP-fucose 
was 1.7 j;M and 38.2 yM for the S100 and P100, respectively. 
The apparent V^ for the S100 was 42.7 pmol fuc/ mg protein/ 
30 min and 122 pmol fuc/ mg protein/ 30 min for the P100. 
As evidenced by the lower apparent K n (22-fold lower), the 
affinity of the S100 fucosyltransferase for GDP-fucose was 
higher than the one from the P100 activity. This accounts 






Figure 4-4. Effect of pH on S100 and P100 fucosyltransferase 
activities in the presence of Tween-20. 

S100 and P100 fractions prepared from vegetative HL250 cells 
were assayed for fucosyltransferase activities in the 
presence of Tween-20 at different pH values (pH values 
determined as described in materials and methods). GDP- 
[ 14 C]fucose concentration was 0.36 jiM; MgCl 2 , 5 mM; Tween-20, 
0.1%. The assay was carried out for 30 min, and 150 vq and 
228 jig of protein were supplied from the S100 and P100, 
respectively. Fucose incorporation was calculated from the 
amount of TCA-precipitable [ 14 C] radioactivity. Results 
expressed as the mean of three determinations i s.e.m. 

Panel A. Effect of pH on S100 fucosyltransferase activity 
in the presence of Tween-20. 

Panel B. Effect of pH on P100 fucosyltransferase activity 
in the presence of Tween-20. 



115 



E 

o 
co 

o 

I— 

a. 

E 
"35 

03 

o 
o 

•*— 

o 

E 

Q. 




PH 



E 
o 

CO 

o 

Q. 
O) 

E 

a> 
to 

o 

o 

»*— 

o 

E 
a. 








T3 
03 <D 




C 




p 

c 

CD 




c 










id to 




•H 






•H 










•o a) s tp 








id 

a 














CM 0) 




>1 






>i 










«S c ^ 




P 






P 






<D 




^oo. 




•H 






■H 






to 




O to ° +J CD 




> 








> 




•». 


(0 






•H 




Ik 




•H 




s 


u 




ft o ?H » 

•H ,i O 4J 




P 




s 




P 




3. 


CD 






u 




3, 




u 






4-1 






(0 








(0 




CN 


to 




TJ+io^d 








r~- 








• 


c 




•o S cn o w 

CD " O Q) 




0) 




• 




CD 




CO 


<0 






to 




rH 




to 




n 


M 






<a 




II 

E 




(0 




II 


P 




O 0S „ C K 




M 






M 




■ 


H 




O -H 

^ CN 

CO • ■ CD • 




0) 




O « 




CD 




« 


10 






4-1 
to 




O 

rH P 




4h 

to 






rH P 







to an 

fucose 

Tween- 

Fucos 

tivity 




c 




cn c 




C 




CO C 


u 






(0 




CD 




(0 




CD 


3 






U 




CD u 




Vh 




CD H 


4-1 






P 




A (0 




P 




A <a 








.H 




P a 




rH 




p ft 


O 






>m 




Oi 




>1 




ft 


O 




•H U £ ' « 




to 




U (0 




to 




u (a 


r-l 
























a, 




2 1o0 




u 




4-1 «v 




u 




4-1 .v 


T3 








3 
4-1 






-^CN 




3 
4-1 






^CN 


c 




p ft v ft <0 








1—1 1 








r— 1 1 


* 




COH 077 




O 

O 




1— • Q) 




O 

O 




CO C 

^<D 


o 




u xijp 




rH 




CD 




r-t 




CD 


o 




•H 4-1 0> P3 




CO 




W Eh 




ft 




CO Eh 


.-i 




P S " 














co 




u 
<o to 2 o) 




c 





> 

4-1 




c 






> 

4-1 


c 




M fi g 4-1 rH 








> 








> O 







4-1 £1 


. 


c 




^ 




c 




\ 






•Hin c a 


e 







r-, CD 









r-. CD 


c 




*P •* ±> 


• 


•H 




CO U 




•H 




CO U 







TJ (BW fll-H 


CD 


P 




^ C 




p 




^ c 


•H 




(1) H O P ft 


• 


(0 




>^ CD 




(0 




s_, CD . 


p 


. 


N P O -H 


to 


u 




to 




M 




. E C 


<0 o 


•H C CD M U 




p 




P CD 




P 




P CD ^ 


V-l cn 


cmuaii) 


+1 


c 




H 




c 




Ohg 


p 


i 


CD U C M 




CD 




M ft 




Q) 




nH ft 


c 


C 


o>e cd >w a to 


U 




ft 









ft O 


<D 


CD 


O to 1 


B 


c 




CD 




c 




CD m 


U 


(1) 


gUCD < 


O 







M x; 









^■C^ 


c 


1 


u c*u 


•H 







(0 P 


• 


u 




to P c 





jC Cn ft 3. En P 









c 






^ -•* 


u 




G 


f0 


CD 




c 


•H 


CD 




O C CD 




4-1 


- -H d) CN 4-1 


c 


to 




U -H 


g 


to 




M-H +J 


CD 


o 


•a >ix: po 


•H 


O 




ft 









ft 


to 




0) *H P *f 


s 


U 




■H >i 





u 




•H >i n 





a) 


P (0 P 


M 


3 




U P 


m 


3 




° H ft 


u 


u 


to > c "0 c 


0) 


4-1 


• 


CD -H 


\ 


4-1 


• 


CD -H 


3 


c 


QJ -H C 3 


p 


1 





M > 


c 


1 





n > a» 


4-1 


0) 


> P (0 


0) 


ft CN 


•rH 


•H 


ft CN 


"1 £ 


1 


to 


n 10 c ex 


Q 


1 


CD P 


CD 


a 


1 


CD P< 


IX 


0) 


(0 "H "» <0 




O 


c 


rH U 


+J 





c 


rH O rH 


Q 


n 


x: e 


<D 




CD 


c^ (d 


O 




CD 


CJi (0 


ts 


a 


H O CD 


CD 


4-1 


CD 


c 


V-l 


4H 


CD 


c -. £ 






J)^OH£ 


M 





t 


•H CD 


ft 





1 


•H CD 04 


4-1 





U -H ro CO P J3 




to to 






CO CQ 


o X 


CD > 


P 


P 




(0 


cr> 


p 




JO CN 




p 


5 j-i <d e 




U 4-1 


to M 


e 


U 4H 


10 U tN 


p 




c £ 


4h 


CD 





CD CD 


\ 


CD 





CD CD rH 


u 


c 


Q) -H 4-1 p M 


O 


4-1 




C MH 


1— 1 


4H 




C MH || 


CD -H 


id 4-i 




4-1 


CD 


<a to 





4-1 


CD 


<a to [ 


4-1 




X3 T3 P H 


c 


U 


U 


x c 


£ 


U 


O 


x c I 


4-1 


to 


CD CD 3 TJ 


(0 




c 


(0 


ft 




C 


(0 > 


w 


CD 


P O «4-t CD 


CD 




CD 


u 






CD 


M 




■H 


e <o p 


E 


• 


to 


• p 


r> 


• 


to 


• p p 




♦J 


IB rH T3 C (0 




< 


CD 


CQ rH 


■ 


u 


CD 


QHC 


• 


•H 


>i CD -H rH 


CD 




U 


>i 


CN 




U 


>i CD 


m 


> 


to -h cd 3 x: 


^H 


a 


rH tO 


"3* 


•-{ 


ft 


rH tO rH 


i 


•H 


m J-l P O 


P 


CD 




CD O 


II 


CD 




CD O (0 


<* + 


CN U U O rH 




C 


01 


c u 




C 


CD 


C O ft 




o 


J 3 ifl M (0 


to 


(0 £ 


(d 3 


B 


(0 X. 


(03ft 


CD 
U 


(0 


s 4-1 a u 


<0 


ft 


p 


ft 4H > 


ft 


P 


ft «w (0 


3 
Cn 























117 





A/S 



A/S 





uilu oe/wejojd 6iu/onj |ouid 



uilu oe/ujeiojd 6w/on} |owd 



118 
for the higher activity of the S100 fraction in the presence 
of detergent at the concentration of GDP-fucose used in most 
assays (0.36 jl/M) , despite the higher V^ of the P100. 

Since the above results were obtained in the presence 
of detergent, I investigated the effect of GDP-fucose 
concentration on the fucosyltransferase activities in the 
intact fractions. It was reasoned that, in the case of the 
P100, it would give us some insight into the overall 
fucosylation process, including uptake of GDP-fucose into 
the vesicles. S100 and P100 fractions were prepared from 
HL250 vegetative cells and assayed for fucosyltransferase 
activity under standard conditions at increasing 
concentrations of GDP-fucose (figure 4-6). An apparent K m 
of 0.44 uM and V of 25.5 pmol fuc/ mg protein/ 30 min was 

~ max 

calculated for the S100. For the P100 an apparent K m of 
28.3 /iM and V^ of 233 pmol fuc/ mg protein/ 30 min was 
determined. 

The fucosyltransferase activities from the S100 and 
P100 differed in the acceptor species that were fucosylated, 
the sensitivity to high pH, divalent cation dependence, and 
apparent affinity for GDP-fucose. These differences in 
enzymatic behavior support a model for separate 
compartmentalization of the two fucosyltransferase 
activities; the S100 activity is free in the cytosol and the 
P100 activity is in a membrane bound organelle. 



d> 






«* 














o 


en 
03 
M 

dl 

4-1 






o 














m 






o 














X. 




T3 0) 


en* 














C 

•H 
d) 
4J 



H 

a 


en 

c 




(fl 0) 


0,+J 

43 1 


4-1 





d) 






di 


03 




o 
o to 


c 




en 

03 






en 

03 


4-> 




^ c 


protein for 
alculated fr 


03 




Sn 


S 

rH 



a 
a 




r4 


rH 
>1 

in 
o 
u 

3 
4H 




and P 
Reactio 


0) 

a 
d) 

4-> 




d) 

4-1 

en 
C 
03 
M 

4-1 




(1) 

4-1 

en 

c 

03 
M 

4-1 



pmol/mg 






o 


en 




i-H 


° .« 




H 


O 




iH 


03 




>1 


o m 




>i 


2 S 
en **> 


O 




CO • 
d> 


T3 




er> 








en 



ft 




c m 


4-1 U 
CO 


0) 




u 


cn 




u 


d) 1' 
■^ 1 






03 


en 




3 


d) " * 




3 


-a 




u 


en 




4-4 


■£ £ 




4-1 


c 




3 


Cn5 


d) 






* > 






+J > 


io 




.p 4-1 

c — ■ 


rH 

0, 




O 

o 


C 




O 

o 


hl-n 


o 




•H U 


°o 


X 




rH 




rH 


° c 


o 




H 


OJ 




en 




ft 


•w ol 


rH 




■0 — ■ 


cn -H 








m 






t-i 


en 




d> 1 


" 4-> 


en 




4-1 


"^ 03 




4-1 


"* 03 






■p ft 


v <0 


4-1 




u 


1—1 Oj 




CJ 


7Z a 


*J 




03 Q 


«n 


rH 




03 


w a 




03 


en a 


u 




C O -H ( 


3 




4-> 


1 — ' 03 




4-> 


•—Jb 


10 




o 


U a 


en 




C 






C 




4-1 




•H 4H 


Cn M 


d) 




•H 


V 




■H 


n, 


c 




4-> S 


OS 






H? £ 






5 as 


■H 




o 


CJ 






c 


> a. 




B 


> a 


C 




03 CO 




• 







>«• 







>m 


o 




4-1 O 




>i 




c 


•^5 




c 


\ . 






•H 


in d> 


4-1 







• 







"oo 


c 




*4-> 


CO 


•H 




•H 


w 




•H 


^cs 


o 




"0 03 4-1 


> 




4-1 


1—1 ii 




4-1 


1—1 II 


■H 




0> H 


CJ 


•H 




03 


-— * a 




03 


— ' B 


4-1 




N 4-1 


3 


4-1 




rH 


M 




u 


M 


03 




•H C 


0) h 


u 




4-> 


4-> 




■p 


4-> 


rH 




C d> 


CJ 


03 




C 


4J 




c 


4-1 


4-> 




CD U 


c 


O 




dl 


H C 




d) 


rH C 


c 




Cn C 


d) . 


•H 




u 


& dl 




U 


a d> 


d> 







CO o 


T3 




c 


M 




c 


(h 


u 




a u 


d) o 


03 







iH 03 




o 


rH 03 


c 







1-1 r-i 


U 




u 


03 CU 




CJ 


o3 a 


o 




x: o^ aft 


I— I 






u a 






u a 


u 




c 




U 




d) 


03 




0) 


o <a 






-•H 


d) CDS 


• 


en 


M 




en 


u 


CD 




n >i-c x: 


i i 


a 





Oj »v 







a •» 


en 




d) M 4-> 4-> 




• 


CJ 


•H >i 




CJ 


•H >i 







4-1 03 




d) 


0) 


3 


CJ 4-> 




3 


CJ 4-> 


u 




CO > 


CHH 


• 


4-1 


d) -H 




4H 


d) -H 


3 




0) 


•H Si 


en 


1 


U > 




1 


V4 > 


4-1 




> 4-> 


4-1 


03 




ft 


•H 




ft 


•H 


1 




rH 03 


c 


4-1 


+i 


Q 


d) 4-1 




Q 


0) 4-1 


ft 




03 


•H C 


•H 




U 


r-l O 




a 


rH U 


Q 




£ 


E -H 


a co 




ty 03 






cnoj 


U 




{-1 


0> 


•H 


C 


4-1 


e 




4H 


c 






d) 4-1 


O 4-> 


u 


o 





•H dl 




o 


•H (!) 


4-1 




H -H 


ro o 


01 


■H 




CO CO 






CO CO 


o 




0) > 


Vh 


u 


4-1 


4-1 


03 




4-1 


03 






£ 


'h ao.id 


CJ 


CO U 


• 


CJ 


CO M 


4-) 




c 





1 


c 


d) 


d) d) 


c 


d) 


d> d) 


u 




Q) -H 4-1 4-1 < 


■H 


4-1 


C 4-1 


•H 


4-1 


C 4-1 


01 




03 





u 


g 


4-1 


03 CO 


a 


4-1 


03 CO 


4-1 


■ 


rQ "O 4J 


Eh 


i-l 


U 


X c 




W 


K C 


4-1 


en 


0) d) 


3 0> 




dl 




03 O 




03 


W 


<l) 


4J 


O 3.4-1 


4-1 


• 


U ro 


• 


r-l 




■H 


a 03 







d) 


• >i 


• 4-> \ 


• >i 


• 4-> 




4-1 


03 >H T3 fN 




•o 


< 4J 


CQ r-l 


B 


U 4J 


Q rH 


• 


■H 


>i d) n 


4-1 




•H 


>1-H 


•H 


>i 


V£> 


> 


O CO 


•H <tf 


C 


d) 


H > 


f-4. CO 


d; 


-H > 


rH CO 


1 


•H 


m o 


M 


3 


d) 


d) -H 


Q) O 


4-1 


d) -H 


d) o • 


*r 


4-) 


cn u 


M TJ 


O 


M 


C 4-> 


C CJ 





C! 4-> 


cue 




CJ 


J 3 


03 C 


ax 


03 CJ 


03 3 


n 


03 CJ 


03 3 -H 


0i 


03 


ac «w 


CJ 03 


03 


4-1 


ft 03 


ft 4-1 


cu 


ft 03 


ft 4-1 g 


3 























120 




a/S 



A/S 








o 




M 


- o 


o 


/ 


, " - 


10 


/ 


. o 

CO 


o 


/ 




*2 


/ 


2 

i 


3. 


/ 


o *■ 


O CD 


J 


CO © 
CO 


• CO 


^ 


o 


CD O 


^^^^ 


o 


O 


^V 


3 


3 






i 


>. 


- o d 


°£ 


^V 


+ Q 


•Q 


Nw 


_ o 


o 


N. 


CM 




O N* 


1 „ 



o 
o 



o 


o 


o 


o 


o 


m 


o 


LO 


o 


in 


C\J 


CM 


T— 


■*— 





inw OG/uiaiojd 6w/onj |oaid 



OZllLu oe/uiejcud 6w/onj |0Lud 



121 

Fucosyltransferase Activity Cannot be Detected In Vitro in 
Ax3 S100 Extracts 

Normal growing cells expressed [ 3 H]fucose metabolically 

labelled FP21 and the main glycopeptide and oligosaccharide 

products released by pronase digestion and A-elimination, 

respectively, were indistinguishable from those derived from 

the mutant HL250 (Chapter III). All the studies reported 

above on the cytosolic fucosyltransferase activity were 

carried out in HL250 because in vitro transfer of fucose 

from GDP- fucose to endogenous acceptors cannot be detected 

in Ax3 S100 fractions by TCA-precipitation, SDS-PAGE 

analysis, or C 18 SepPak fractionation (see Materials and 

Methods for description of methods). A plausible 

explanation for the lack of activity in the Ax3 would be 

that the S100 from Ax3 contained an inhibitor for the 

activity. To investigate this possibility, I performed 

several mixing experiments. Vegetative Ax3 and HL250 cells 

were harvested, filter-lysed and fractionated into an S100 

and P100. The S100 fractions were assayed individually or 

mixed in different ratios before assaying for 

fucosyltransferase activity. The fractions were used intact 

or desalted prior to assay (table 4-4). There was no 

evidence for an inhibitor since experiments in which S100 

fractions from Ax3 and HL250 were mixed in different ratios 

showed activity commensurate to the HL250 contribution 

(table 4-4). Dilution of labelled GDP-fucose with 

endogenous unlabelled GDP-fucose is not an explanation 



122 



c 
o 

•H 

p 
u 

P 
14-1 

o 
o 

<-t 

C/3 

m 

< 



p 



p 
u 

<0 
CD 

to 

P 
CD 

<« 

cn 
c 
<c 
u 
p 
^ 

>1 

to 
o 
u 

fa 



I 
CD 

i-H 

£1 
(0 



T3 
CD 
+J 
.H 

IT) 

cn 

0) 

Q 



P 



p 



p 
cj 

ro 



S 

a. 
Q 



o 

p 

•H 
> 

•H 
P 

u 

(0 



p CD 
u p. 
•a 

p 

c 



c 
o 

•H 

p 

3 
£1 

•H 

P 
P 
c 
o 
u 



ro 

< 



OOO 
ID CN 00 

O r-i O O O 



tH o in 00 r-» 

o o ro o r- 

O rH o o o 
v 



in 00 

Hmooo\ 
r» vo ih o 

00 CN CT> CN CN 



ih o vo >«f r- 

oointNh 



O <H O O O 
V 



cn o in 
^ ro in cn 

OOOfO* 
00 r-i iH ^l< i-H 



OHHH* 



HOH^H 



>i 
■P ? 

■H P 



P O 
CJ O 

id 

p 



<D 
Cn 

a 

P 
CD 
> 
(0 

0) 

p 

CD 
P 
(0 

cn 

p 

3 

CO 

0) 

OS 



u 

(0 



c 
o 



p 

(0 



1) -J-CN 



M-l 0) o 



in 






P H 

a to 
p 



T3 
0) 

P 

H 

to 
cn 
<D 
•d 

M 



P 

u 

(0 

p 

c 



>1 

'■d 

CD 

p 

.Q 
■H 

P 
P 

C 

o 





T3 

CD 



T3 
O (0 



10 



CN 



CD 
E 

3 



CD 
I* 

(0 . 
03 C 

2H 

<fl g 

•0 

c o . 

(0 ro 
o l, H 

O H) 
in cn 

CN 03 

J (0 
X 

'T3 

P 
fd 
TJ 
c 
id 
p 
cn 



" P 
* P 

o 



o 
> 

o 

p 



T3 
CD 
X 

■H 

e 

cn 

<o 

o 
o 

i-H 
C/3 

ro 

< 



CD 
P 
CD 

cn 

CD 

s 

cn 



CD 

A 
P 

P 
o 



•H 
P 

f0 
H 

CD 
P 

CD 

C 

o 
n 

cn 

(0 
EH 

c 

•H 

•H 

S 

■d 

c 

>< 
a 

cn 
cn 
cc 



H P 



c 
o 

■H 

P 
(0 

p 
p 
c 

CD 
CJ 

C 

o 

CJ 

id 

p 

cn 
id 



ar! 



U3 



c o 

3 o 



a 

in 

00 

P 
co 

c 
o 

•H 

p 

(0 

p 

p 

c 

CD 

u 

c 
o 

CJ 



03 



CD 



03 
(0 ro 

*i 

,P«> 



in 



> 

■H 

P 


(0 



CD 
P 
O 
P 
Q, 

m 
X 

.< 



in 



c 
o 

■H 

p 
u 

p 






0) 
cn 

3 



P 
•H 
> 
•H 

P 
U 
(0 



in 

CN 

PI 

X 

Cn 

C 
•H 

cn 

3 

■o 

CD 
P 
rd 

—I 

3 

<0 o 

>i 3 
P .Q 



C" 

a 

vo 

p 
ta 

o 
in 

CN 

P3 

K 

T3 

C 
(0 



•h -h a 



CD 

p 
o 
p 



Cn 

a 



123 
either because activity was not detected in Ax3 after 
desalting through a P-2 column, while the HL250 S100 
retained activity (table 4-4). Another explanation for the 
lack of activity in the Ax3 S100 is that FP21 from Ax3 was 
quantitatively fucosylated in vivo, leaving no acceptor 
sites for the reaction in vitro. Evidence using purified 
FP21 from HL250 supports this conclusion (see next section). 

Fucosyltransferase Activity Is Detected in Ax3 S100 Fraction 
Upon Addition of Mutant FP21 

The absence of cytosolic fucosyltransferase activity in 
Ax3 S100 extracts could be explained as a result of 
quantitative fucosylation of FP21 in the living cell. It 
was reasoned that if this model was correct, then addition 
of mutant FP21 to Ax3 extracts would lead to incorporation 
into FP21. FP21 was trace-labelled in vitro using GDP- 
[ 14 C]fucose, and partially purified by ammonium sulfate 
precipitation, QAE-ion exchange chromatography, and HPLC gel 
filtration. Fractions from the gel filtration step were 
counted and examined by SDS-PAGE and those that contained 
FP21 were pooled, brought to dryness, dissolved in water, 
and added to Ax3 S100 extract. In vitro fucosylation was 
determined as [ 3 H] incorporated into TCA-insoluble material 
in the presence of GDP-[ 3 H]fucose. Since FP21 was trace- 
labeled with [ 14 C]fucose, the relative amount of the 
acceptor added was estimated from [ 14 C] dpm. West et al. 
(unpublished results) showed that incorporation was 



124 
proportional to the amount of [ 14 C] radioactivity added. 
Since this study did not analyze the in vitro fucosylated 
species by SDS-PAGE, it cannot be concluded that Ax3 was 
able to fucosylate FP21. To investigate which MW species 
served as acceptor for the Ax3 cytosolic fucosyltransferase, 
the experiment was repeated using a new batch of partially 
purified [ 14 C]FP21. In vitro labelled FP21 eluted in 
consecutive fractions 20 and 21 during HPLC gel filtration 
chromatography, as confirmed by SDS-PAGE. Ax3 S100 was 
added to an aliguot of fraction 21 that had previously been 
dried on the bottom of the assay tube and assayed for 
fucosyltransferase activity in the presence of GDP- 
[ 3 H]fucose and Mg ++ . The reaction was stopped by boiling in 
sample electrophoresis buffer, resolved by SDS-PAGE, and the 
gel sliced and counted. Figure 4-7 shows the comigration on 
SDS-PAGE of the in vitro [ 3 H] label resulting from 
fucosylation by Ax3 with the trace [ 14 C] labelled FP21 from 
fraction 21. There was no [ 3 H] radioactivity incorporation 
into any MW species when the Ax3 S100 fraction was incubated 
in the absence of purified HL250 FP21. In conclusion, Ax3 
S100 has a cytosolic fucosyltransferase activity that 
utilizes the same acceptor as the mutant cytosolic 
fucosyltransferase. 



Figure 4-7. Fucosylation of mutant FP21 by Ax3 S100 fraction. 

HL250 amoebae were harvested, homogenized, and fractionated 
into S100 and P100 fractions. Unlabeled HL250 S100 was 
mixed with an aliquot of in vitro [ 14 C]fucosylated HL250 
S100. FP21 was purified from the S100 fraction by (NH 4 )S0 2 
precipitation, QAE-ion exchange chromatography, and HPLC gel 
filtration (described in detail in Materials and Methods). 
[ 14 C]FP21 eluted in fractions 20 and 21 of the HPLC gel 
filtration chromatography, as confirmed by SDS-PAGE. Ax3 
S100 was added to an aliquot of fraction 21 that was 
previously dried down in the bottom of an assay tube in a 
vacuum centrifuge to serve as acceptor in the in vitro Ax3 
S100 fucosyltransf erase reaction. The reaction mixture 
contained 0.15 jiM GDP-[ 3 H] fucose, 5 mM MgCl 2 , 349 vq of Ax3 
S100 protein, was incubated for 60 min and the reaction 
stopped by boiling in SDS/fi-mercaptoethanol electrophoresis 
buffer. Sample was resolved on a 15% SDS-polyacrylamide 3 
gel, which was cut into 0.5 cm slices and counted. No [ H] 
radioactivity was incorporated into any MW species when the 
wild type Ax3 S100 fraction was incubated in the absence of 
added FP21 from mutant source (not shown). Open circles, 
[ 14 C] label derived from in vitro labelled purified FP21 
from HL250; closed circles, [ 3 H] radioactivity from in vitro 
fucosylation by the Ax3 S100 fraction. 



126 



100 



80- 



60- 



40- 



20- 




127 

Cytosolic Fucosyltransferase Preferentially Fucosylated a 
Type I Acceptor 

The size of the fucose-containing oligosaccharide in 

FP21 (determined in Chapter III to be 4.8 glucose units) 

implies there is more than one sugar residue, so the 

acceptor site on FP21 may be another sugar. As a first step 

to determine whether the cytosolic fucosyltransferase could 

fucosylate model acceptor analogs utilized by known 

fucosyltransf erases, the S100 from Ax3 and mutant origin 

were screened for activity towards hydrophobic model 

acceptors. The incorporation of radioactivity into 

synthetic sugar acceptors that contained 8-methoxy 

carbonyloctyl, or methyl nonanoate, [CH 3 (CH 2 ) 7 COOCH 3 , 

referred to as R throughout the text] as the hydrophobic 

tail by the S100 was determined by the C 18 Sep-Pak method, 

which employs a hydrophobic interaction column. Unreacted 

GDP-fucose does not interact with the column, eluting in the 

water wash while the glycolipid acceptor is eluted from the 

column with methanol. As shown in table 4-5, only the type 

I acceptor analog (known as lacto-N-biose I or 

galfil,3GlcNAcfi-R) sustained activity in the Ax3 S100. In 

contrast, type II (known as N-acetyllactosamine or galfll,4- 

GlcNAc-R) and fl-gal-R were not suitable acceptors (see 

figure 4-8 for structures). The mutant S100 was also active 

with galfll,3GlcNAcfi-R, but only one tenth as active (on a 

per protein basis) as Ax3. 



128 



Table 4-5. Utilization of 8-methoxycarbonyloctyl synthetic 
acceptors by cytosolic fucosyltransferase activity from Ax3 
and HL250. 

pmol fucose/mq/h 
substrate concentration (mM) Ax 3 HL250 

fi-gal-R 1 und - n - d * 

galfil,3GlcNAcfi-R 0.15 0.38 0.039 

galfll,4GlcNAcfl-R 0.15 und. n.d. 



Incorporation was determined by the C 18 Sep-Pak assay (see 
Materials and Methods) . Experiment performed by CM. West and 
is the result of one determination, und, undetectable; n.d., 
not determined; -R is - ( CH 2 ) 8 COOCH 3 ; GDP-[ HJfucose 
concentration, 0.15 £/M. 






Figure 4-8. Haworth projections of the structures of the 
synthetic glycolipid acceptors. 



130 



CH,HH 




CH,.0H 



CH 2 0H 




NHCXH, 



H OH 

2. galfl(1,3)GlcNAcD-R 



CH.-.CH 




H OH 

3. ga1B(l,4)GlcNAcB-R 



H NHCOCH, 



R= (CH 2 ) 8 C00CH3 



131 

8-methoxycarbonyloctyl synthetic acceptors are not 
available commercially, so I investigated the possibility 
that other hydrophobic glycosides, which can be readily 
obtained from commercial sources, would serve as acceptors. 
Some of these phenyl derivatives have been shown by others 
to be suitable acceptors for a fucosyltransf erase (Potvin, 
et al., 1990; Palcic et al., 1988). S100 fractions from 
HL250 and Ax3 were assayed for fucosyltransferase activity 
in the presence of p-nitro-phenyl glycoside derivatives; 
fucosylation of endogenous substrates was monitored by TCA- 
precipitation, and fucosylation of p-nitro-phenyl glycosides 
by the Sep-Pak method (table 4-6). Inhibition of 
incorporation of radioactivity into FP21 in HL250 was 
examined by TCA precipitation of endogenous acceptors. 
Millimolar concentrations of these compounds failed to 
inhibit significantly incorporation. Likewise, none of the 
p-nitro-phenyl glycosides served as acceptor for the mutant 
nor the Ax3 cytosolic fucosyltransferase activity when 
assayed by the C 18 Sep-Pak method. Thus it is concluded, 
that from the acceptor candidates examined, only 
galJil,3GlcNAcfi-R is a suitable acceptor under the conditions 
used. 

The activity responsible for fucosylation of the type I 
acceptor analog in Ax3 was examined by varying the 
concentration of acceptor or the concentration of nucleotide 
sugar donor. The type I acceptor analog was fucosylated 



132 



u 




^ 










T3 


cn •» 







<D 










C rH CD 


4-1 


m 


u H 










(0 


ra cn 

•rH o 


cn 


x 


M 










>■ 


U u 


M 


< 


f0 










■P 


CD 3 







cu 






. 




•H 


+J 4-1 


■P 


s 


a 


m *r TJ 


o 


> 


CO r-. 


cu 


cu 


a) 


V 




• rH rH 


rH 


•H 


S U 


0) 


Q 


CO 






c 




■P 




u 




■ — ■■ 










u 


u 














<fl 


S i 


ra 
















cn Q. 


tn 




CO 










in T3 u 


tn 
<d 
■o 

■H 

W 

CJ 
>i 
i— 1 


o 
in 

CN 

X 


<0 

a 


n 


H 


CM VO rH CO 


"3* 


(0 O s 
Vh JS 3 
CD -P 
<« CD cn 

cn E co 

G „ • 

(0 So 

u u 
■p ,v 

rH*r-l 


2 

Q 


<D 

cn 


V 


CN 


HCNfOfl 


CN 


cn * 














>1 


(0 >i 


w o 














cn 


c^ c 


rH° 














o 


Cu CD 


*£ 

















CD £ 


C« 














3 


cn a 


^5 


o 












4-i 


M » 


IT) 
CN 


^^^ 










M 


Si 


-nitro- 
and 


h4 


< 


m 


UO 


O VO CN CT> 


T 


O 


c ••* 


X 


u 


o 


CT> 


cn vo n m 


m 


4-1 


O 73 




E-i 


o 


a\ 


o o o o 


<-4 




•■H CD 


s 




r-t 




rH rH rH i-H 


rH 


•o 


■P c 


ex 

Q 












CD 


(0 -H 

■p e 


a 10 

CN 














CO 

cn 


•H M 
(X CD . 


4-1 1-3 














cn 


•H +J >i 


K 














A3 


CJ CD CO 
















CD TJ CO 


+J -H 














CD 

u 


U , CO 

a+J CO 

1 2 


•H 

^ >1 














CD 
5 


•H +J 


C 












U k -H 


XI -H 















o 
in 

CN 

X 


& 1 . s 


suita 
activ 


■H 

-P 

■P 

c 






s 


iiii 


| 


r< rH G 
•H II '? 


CD (D 


CD 












•a 


£ {0 


CJ 






O 


o o o o 


o 


c 


3 -2 

^ -H tJI 


■P (0 


c 






i-H 


rH rH l-H rH 


rH 


<C 


, n 


o 














« 0) 


u 












m 


«« 










0) 




X 


.2 S 








CO 


(0 

0) o 




< 








cn 


cn cj cj -P 




B 



u 


c « 3 - 


3^ 











< < U 




o 5 








CJ 


U22(B 




CO ~ «5 








3 


3 U U r-\ CJ 










rH 


HHH I0H 














cn ct>u u cn U 

1 1 1 1 1 1 
Q Q Q Q Q Q 
1 1 I 1 1 1 


CD 


cn 
B 

O 


H SH 

o o •- 

M CN 


4-1 








0c5 cs <S cc +J 


•H 


O cn i 


. 








I 


1 1 1 1 1 


(0 


■P 


u -o S 


lO U 


4J 






8 S S S S S+J 


U 


C g 


1 -H 


c 






1 


1 1 1 I 1 


CD 


(0 


■H-3 | 


^< rH 


CD 




rH 





O o 


CJ 


CD aj Eh 





S 




o 


u 


S-i M S-l M M 


<o 


4-1 


V (0 


■P 




n 


■p 


+J .p 4J +J -P 


CO 




cn s 


rH 


(fl 




-p 


•H 


•H -H >H -H -H 


u 


O 


<*> 


.a 4-) 


CD 




c 


c 


c c c c c 


■P 


O 


CJ T3 -H 


(0 >i 


M 







1 


1 1 1 1 1 


CD 


<-4 


3 C • 


Eh CJ 


-P 







a ch a a & a -p 


C/3 "+H CO O 



133 
with an apparent K m of approximately 1 mM for the acceptor 
and 1.6 jjM for GDP-fucose (CM. West, unpublished results). 
The similarity in the apparent K m for GDP-fucose for the 
type I and FP21 fucosyl transferase suggested that the same 
enzyme may be responsible for both reactions. The fact that 
HL250 was able to fucosylate so poorly the analog when 
compared to Ax3, suggested that the availability of 
endogenous substrate (FP21) inhibited incorporation into the 
synthetic acceptor, and supported the idea of the same 
enzyme fucosylating both substrates. The notion was further 
reinforced by reduction of type I analog fucosylation in Ax3 
S100 extracts by purified FP21. Fractions 20 and 21 from 
the HPLC gel filtration chromatograph (see section above on 
reconstitution of Ax3 fucosyltransferase activity by 
purified FP21) reduced incorporation of radioactivity into 
type I acceptor (table 4-7). Even though reduction was not 
strictly proportional, it was evident that it increased with 
increasing amounts of FP21. Taken together the results of 
this section and the preceding one, it appears that Ax3 
possesses a fucosyltransferase activity in the cytosol 
capable of fucosylating FP21 and the type I analog acceptor. 

Cytosolic Fucosyltransferase Activity is Present in 
Migrating Slug Stage Cells 

Cytosolic FP21 was detectable by metabolic labelling in 

slug stage HL250 cells (Chapter III). Reasoning that a 

fucosyltransferase responsible for its modification would be 



134 



Table 4-7. Reduction of fucosylation of acceptor type I 
analog by purified FP21. 

fraction relative amount r 3 H"|fucose incorporated 

added (dpm/mq protein/h) 

499 

20 lx 387 

20 4x 318 

21 lx 436 
21 4x <20 



Transfer of [ 3 H] from GDP-[ H]fucose into 4 /ig (0.145 mM) of 
type I acceptor analog by Ax3 S100 was measured using the C 18 
Sep-Pak method (see Materials and Methods). Data are the 
results of one determination. 349 jig protein of Ax3 S100; 
GDP-[ 3 H]fucose concentration, 0.15 juM; 60 min assay. 



135 
present at this stage of development, I assayed developing 
cells for their ability to incorporate [ 14 C] from GDP- 
[ 14 C]fucose into endogenous acceptors. HL250 cells were 
plated for development and at slug stage harvested, 
disaggregated, fractionated into an S100 and P100, and 
assayed for fucosyltransferase activity as described in 
Materials and Methods. For comparison, fractions from Ax3 
slugs were assayed (table 4-8). As seen with amoebae cells, 
HL250 S100 was active whereas the Ax3 S100 did not 
incorporate radioactivity. On the other hand, the P100 was 
active in both strains. Thus, it seems that the cytosolic 
fucosyltransferase is not restricted to the growth phase and 
is present in developing cells. 

Discussion 
The presence of FP21 in the cytosol suggested that a 
fucosyltransferase might also be located in the cytosol. An 
S100 fucosyltransferase activity was detected which was both 
time- and protein concentration-dependent. The activity was 
strictly divalent cation dependent. Incorporation of 
radioactivity was sensitive to temperature, certain 
detergents, and ethanol. A variety of sugars and sugar- 
derivatives failed to inhibit activity, except GDP-fi-fucose, 
which inhibited in a dose-responsive manner. The activity 
could not be sedimented by higher centrifugation force in 
the presence of an extensive list of protease inhibitors. 



136 



Table 4-8. In vitro fucosyltransf erase activity of HL250 and 
Ax3 slug extracts. 

pmol fucose/mq protein/30 min 
strain S100 P100 

HL250 40.6 36.3 

Ax3 <0.1 30.7 

Normal and mutant amoebae were allowed to develop, harvested, 
disaggregated, f ilter-lysed, and fractionated into an S100 and 
P100. Intact fractions were assayed for fucosyltransf erase 
activity in the presence of 0.36 juM GDP-[ C]fucose, 5 mM 
MgCl , 12-48 vq of protein, for 30 min. Results are the 
average of two determinations. 



137 
The endogenous acceptor utilized by the S100 
fucosyltransferase was a protein which comigrated with FP21 
by SDS-PAGE. I compared the acceptor for the in vitro 
fucosyltransferase reaction with metabolically labelled FP21 
from Ax3 cells by SDS-PAGE. There was one main radioactive 
peak, revealing in vivo and in vitro fucosylated acceptors 
with the same mobility on polyacrylamide gels. These 
results suggested that a cytosolic fucosyltransferase 
existed that utilized FP21 as its primary acceptor species 
in vitro, and may be responsible for fucosylation of FP21. 

To investigate the origin of the S100 
fucosyltransferase, I compared it to the bulk P100 
fucosyltransferase activity, since the cytosol is the 
default location of lumenal enzymes released by rupture of 
vesicles. If both activities were indeed different, I 
expected to detect enzymatic differences. Initially, I 
examined the SDS-PAGE profiles of in vitro fucosylated 
acceptors and found they were very similar to those obtained 
from metabolic labelling. Incorporation by endogenous 
acceptors was at the 21 kD MW position for the S100, and in 
the P100 radioactivity migrated as two separate, broad 
peaks . 

In order to compare directly the soluble and the 
sedimentable activities, I assayed the S100 and P100 in the 
presence of detergent to overcome any differences in 
accessibility for GDP-fucose by the fucosyltransf erases . I 



138 
determined that the S100 fraction was dependent on divalent 
cations, while the P100 was active in the absence of cations 
and in the presence of the chelator EDTA. The activities in 
both fractions were maximal at a similar pH range, but the 
cytosolic fucosyltransferase was more sensitive to higher pH 
than the P100 fucosyltransferase activity. 
Glycosyltransferase activities have commonly been found to 
be dependent on the presence of divalent cations. In the 
case of fucosyltransf erases, however, there are precedents 
for al,2, al,3, and crl,3/l,4 fucosyltransf erases which are 
active in the absence of cations, and are either stimulated 
or inhibited by different cations (Beyer and Hill, 1980; 
Campbell and Stanley, 1984; Foster et al.,1991; Stroup et 
al., 1990; Zatz and Barondes, 1971). 

The apparent affinity for GDP-fucose differed greatly 
for S100 and P100 activities. The S100 fucosyltransferase 
activity had a higher affinity for GDP-fucose than the P100 
activity when both were assayed in the presence of Tween-20. 
The lower apparent K for the cytosolic fucosyltransferase 
explained why activity is higher in the S100 at the low 
concentration of GDP-fucose used in most assays, 0.36 ^M. 
At 0.36 pM the concentration of GDP-fucose was near its 
apparent K m for the S100 fucosyltransferase (1.7 jjM) , but 
well below the apparent K n for the P100 enzyme (38.2 /jM) . 
The dependence on GDP-fucose concentration was also examined 
in the intact fractions to gain some insight into the 



139 
overall fucosylation process in the P100 fraction, including 
transport into the intact vesicles. The apparent Michaelis 
constants for the P100 activity in the presence of Tween-20 
and in the intact fraction were 38.2 jl/M and 28.3 jjM, 
respectively. The similarity of the apparent K m values 
suggested that the GDP-fucose transporter in the P100 
vesicles had a similar or lower K m relative to that of the 
bulk P100 fucosyltransferase activity, since if it had a 
much higher apparent K m , GDP-fucose transport would have 
been rate limiting. The GDP-fucose transporter from rat 
liver Golgi-enriched vesicles has an apparent K m of 7.5 jiM 
(Sommers and Hirschberg, 1982). The apparent K m for the 
cytosolic fucosyltransferase is relatively low compared to 
that of the bulk P100 activity. Though the relative 
concentrations of GDP-fucose in the cytosol and vesicles are 
not known, vesicles have the ability to concentrate GDP- 
fucose relative to the outside (Perez and Hirschberg, 1986). 
Thus it is not unreasonable to predict that a cytosolic 
fucosyltransferase would have a higher affinity for GDP- 
fucose since the concentration of GDP-fucose is probably 
lower in the cytosol than in the vesicles. 

The studies described in this chapter concerning the 
P100, characterized the bulk activity in the fraction and 
cannot differentiate among different fucosyltransferases 
that may be present. The fucosyltransferase activity in the 
P100 may be a product of different fucosyltransferases with 



140 
different specificities. This may be the case in 
Dictyostelium because, even though fucosyltransf erases have 
not been well characterized in this organism, various 
fucosyltransferases have been localized to microsomes in 
other eukaryotes (Hirschberg and Snider, 1987; Kornfeld and 
Kornfeld, 1985). The fact that I was able to differentiate 
the bulk activity in the P100 from the cytosolic 
fucosyltransf erase supported the idea that the 
fucosyltransferase in the S100 is unrelated to the P100 
activity and thus endogenous to the cytosol . 

However, my observations do not rule out other 
possibilities. For example, the cytosolic 

fucosyltransferase could have derived from vesicles but was 
preferentially lost during isolation and the remaining 
enzyme, though with distinct properties from the majority of 
the P100 activity, is in the minority. The inability of 
EDTA to inhibit activity further when compared to no 
addition of divalent cations to the P100, may mean that the 
enzyme does not need cations at all. Conversely, since the 
bulk activity in the P100 is stimulated by cations, it is 
possible that the activity retains tightly bound cations 
which EDTA cannot remove. Another possibility is that the 
acceptor, FP21, is not present in the P100, either due to a 
cytosolic compartmentalization, or to leakage from the 
vesicles. A definitive confirmation that the cytosolic 
fucosyltransferase is different from any fucosyltransferase 



141 
activity in the P100 will require characterization of the 
purified fucosyltransf erases from the S100 and P100. 

The results obtained from my investigation are based on 
biochemical evidence in which a soluble fucosyltransferase 
partitioned with the cytosol. Other investigators have 
identified glycosylated proteins in the cytosol and/or 
nucleus and have searched for an enzyme responsible for the 
addition of the sugar (Haltiwanger et al., 1990). Their 
biochemical studies showed that an activity capable of 
adding GlcNAc to protein was recovered in both the soluble 
and membrane fractions (Haltiwanger et al., 1990). However, 
they showed that the membrane-associated activity was 
releasable by high salt treatment and was oriented towards 
the cytosol, not the lumen of the vesicles. Thus, it is 
possible that a fraction of this newly discovered cytosolic 
fucosyltransferase stayed associated with vesicles but since 
it was in a minority, remained masked by other P100 
fucosyltransf erases. As more synthetic acceptors become 
available, latency experiments in the presence and absence 
of detergent can be done to address this question. 
Alternatively, it is possible that a fucosyltransferase with 
enzymatic properties similar to the cytosolic 
fucosyltransferase is present in the lumen of P100 vesicles. 
Still this will not contradict my findings and will imply 
that there are two similar enzymes that reside in distinct 
compartments, as has been reported for another enzyme (Lewin 



142 
et al., 1990). In any event, I interpret the data presented 
as evidence for a fucosyl transferase in the cytosol of 
Dictyostelium discoideum. 

The fact that Ax3 produced fucosylated FP21 suggested 
that, as it occurred in the mutant, the normal strain may 
have a cytosolic fucosyltransferase responsible for FP21 
fucosylation. However, while activity was not detectable in 
Ax3 S100 fraction, it could be reconstituted by addition of 
mutant FP21, indicating that Ax3 possessed a cytosolic 
fucosyltransferase eguivalent to the mutant 
fucosyltransferase . 

In order to characterize the fucosyl linkage catalyzed 
by the cytosolic fucosyltransferase, several acceptors were 
used. Activity with synthetic acceptors was about an order 
of magnitude higher for the Ax3 extract, which may be 
attributed to competitive inhibition by the unfucosylated 
FP21 in the mutant. Of those tested, the only suitable 
acceptor was found to be a type I analog, 8- 
methoxycarbonyloctyl gal/il,3GlcNAcfi. Since the type II 
analog [ 8-methoxycarbonyloctyl galJ31,4GlcNAcfi] did not work 
as acceptor, it appears that the cytosolic 
fucosyltransferase may be an al,4fucosyltransf erase. 

The cytosolic fucosyltransferase preferentially 
recognized a type I analog, suggesting it was an 
al, 4 fucosyl trans f erase that lacked crl,3 activity. This 
activity would differ from other al,4fucosyltransf erase 



143 
described, which exhibit al,3 activity as well (Kukowska- 
Latallo et al, 1990; Stroup et al., 1990). However, there 
are some limitations to the studies employing synthetic 
acceptors. To conserve synthetic acceptors, which were not 
commercially available, the concentration of the acceptors 
was well below the K m (0.145 mM, while the apparent K b was 
determined to be approximately 1 mM) . The possibility still 
exists that the enzyme is able to use 8-methoxycarbonyloctyl 
galfll,4GlcNAcfi as acceptor, but will only be evident at 
higher concentrations. Tentatively, an al,4 specificity is 
being assigned to the cytosolic fucosyltransferase, but 
definitive proof will reguire characterization of the enzyme 
purified to homogeneity. 

Finally, slug stage extracts were examined for 
fucosyltransferase activity, because it was found by 
metabolic labelling experiments in Chapter III that a 
fucosylated protein of 21 kD fractionated with the S100. 
The S100 and P100 fractions from HL250 had considerable 
activity, but from the Ax3 fractions only the P100 showed 
activity, consistent with the results from vegetative cells. 
The detection of a cytosolic fucosyltransferase in slug- 
stage cell extracts is consistent with their ability to 
fucosylate FP21 in vivo as determined by metabolic 
labelling. The apparent absence of activity in Ax3 cells 
indicated that, as found for vegetative stage cells, FP21 
was guantitatively fucosylated. 



CHAPTER V 
SUMMARY AND CONCLUSIONS 



Summary of Results 
Fucosylation has generally been regarded as a 
modification restricted to the secretory compartment, 
however, there is evidence of fucosylated macromolecules in 
the nucleus and cytosol (see Chapter I). In the present 
study, I identified a novel fucosylation pathway in the 
cytosol of Dictyostelium discoideum. In the next three 
paragraphs a short summary is presented of the results 
reported in this dissertation, followed by a proposed model 
of fucosylation in the cytosol. 

In chapter II the mutant HL250 was characterized as a 
conditional fucosylation mutant. The results are summarized 
as follows: 1) Spores and vegetative cells from the mutant 
strain contained negligible amounts of macromolecular- 
associated and total cell fucose when compared to the normal 
strain, Ax3, as determined chemically in acid hydrolysates . 
2) The phenotype was conditional to growth in the absence of 
fucose. When vegetative cells were grown in fucose- 
supplemented media, they expressed macromolecular fucose 
conjugates. The fucose specific activity of the medium was 
not diluted relative to the intracellular fucose. 3) 

144 



145 
Mutant extracts were incapable of carrying out the 
conversion of GDP-mannose to GDP-fucose in vitro. In other 
organisms, this pathway is the sole pathway of GDP-fucose 
synthesis in the absence of extracellular fucose. The low 
fucose biochemical phenotype can be explained by the model 
that the conversion pathway is defective. HL250 cells and 
extracts in vitro can still fucosylate, showing that GDP- 
fucose transport and fucosyltransferase(s) are still active. 
Although the possibility remains that there are other 
genetic defects in this mutagenized strain, there is no 
reason to suspect that other genes of the fucosylation 
pathway have been affected. 

After determining that the source of macromolecular 
fucose in HL250 grown in normal medium was derived from 
extracellular fucose, I examined the compartmentalization of 
fucosylation. The results of the experiments described in 
Chapter III show the existence of a fucosylated protein in 
the cytosol and are summarized as follows: 1) The major 
fucosylated species in the S100 is FP21. It is present in 
both Ax3 and HL250. 2) Analysis of FP21 revealed that the 
oligosaccharide in FP21 was O-linked with a size of 4.8 
glucose units. 3) FP21 appears to be endogenous to the 
cytosol, and not derived from a sedimentable compartment 
during preparation of the extracts. 4) Glycopeptides 
released from FP21 by pronase digestion differ from 21 kD MW 



146 
PlOO-derived glycopeptides, which reinforced the notion that 
contaminating P100 material was not the source of FP21. 

The presence of a cytosolic fucosylated protein 
suggested the existence of a cytosolic fucosylation pathway. 
In vitro analysis of subcellular fractions led to the 
detection of a fucosyltransferase activity in the cytosol . 
The results of this investigation described in Chapter IV 
are summarized as follows: 1) Using a fucosylation assay 
dependent on endogenous acceptor substrates, I detected 
fucosyltransferase activity in cytosolic and vesicular 
fractions. 2) Activities from S100 and P100 fractions 
differed in the acceptor species fucosylated, their 
sensitivities to alkaline pH and divalent cations, and 
affinities for GDP-fucose, as evidenced by differences in 
apparent K . I consider these results to be an indication 
that the S100 fucosyltransferase did not arise from vesicles 
by rupturing during cell fractionation. 3) The cytosolic 
fucosyltransferase activity was absolutely dependent on 
availability of a non-fucosylated acceptor. Accordingly, in 
vitro cytosolic fucosylation could be detected in mutant 
extracts, but not in Ax3 fractions. However, cytosolic 
fucosyltransferase activity was reconstituted in Ax3 
fractions by addition of purified mutant FP21. 4) A 
fucosyltransferase activity was detected in the S100 with 
the use of synthetic hydrophobic acceptors. Based on the 
utilization of these acceptors, the activity was determined 



147 
to be an al,4fucosyltransf erase lacking crl,3 activity. 5) 
Fucosylation of the type I acceptor analog (galfll,3GlcNAcfl- 
8-methoxycarbonyloctyl) was inhibited by addition of 
purified FP21, suggesting the same activity was responsible 
for fucosylation of both molecules. 

Based on the results obtained in my studies, I propose 
a model for fucosylation in Dictyostelium, acknowledging the 
existence of a fucosyltransferase in the cytosol that 
fucosylates a cytosolic protein, FP21. There are 
fucosyltransf erases in vesicles and in the cytosol; the 
preferential acceptor for the cytosolic fucosyltransferase 
is FP21. This model is appealing because all of the 
elements necessary for fucosylation, biosynthesis of GDP- 
fucose, a fucosyltransferase, and the acceptor, 
compartmentalize in the cytosol. The model also concurs 
with emerging views of glycosylation in the cytosol (Hart et 
al., 1989a; Hart et al., 1989b). Initially I showed that 
Dictyostelium possesses a GDP-fucose conversion pathway 
similar to that reported earlier for other organisms 
(Kornfeld and Ginsburg, 1966; Liao and Barber, 1971; Ripka 
et al., 1986). It was shown that Dictyostelium can convert 
GDP-mannose into GDP-fucose, and that when this biosynthetic 
pathway is defective, GDP-fucose is formed from fucose 
supplied in the extracellular medium by the salvage pathway. 
This is the first time evidence has been presented that 
suggests Dictyostelium has GDP-fucose biosynthetic pathways 



148 
similar to those found in bacteria (Kornfeld and Ginsburg, 
1966), a higher plant (Liao and Barber, 1971), and mammalian 
cells (Ripka et al . , 1986; Reitman et al . , 1980). 

However, this model is not the only one that could 
account for the data obtained during the course of my 
investigation. Alternatively, the presence of FP21 in the 
cytosol could be explained by fucosylation in vesicles and 
rapid posttranslational transport to the cytosol. The 
absence of FP21 and FP21-like glycopeptides in the vesicular 
fraction was interpreted earlier as evidence for the absence 
of FP21 in the P100. However, it does not rule out the 
possibility that FP21 was fucosylated in vesicles and soon 
thereafter transported back into the cytosol, but was not 
detected because it did not accumulate in the P100. The 
presence of a fucosyltransferase in the cytosol would then 
be accounted by leakage from the vesicular fraction. 
Clearly, this model must then explain the export of FP21 
into the cytosol by novel and unknown mechanisms. Another 
model that would account for my results is that both FP21 
and the fucosyltransferase detected in the S100 leaked into 
the supernatant during fractionation. Since known vesicular 
markers were shown to remain in the P100, this model would 
require FP21, from all fucoconjugates in the P100, to be 
released preferentially. It would also require the leakage 
of a fucosyltransferase capable of fucosylating FP21. 
Nevertheless, in order to distinguish between the model 



149 
proposed and the other possible models, additional studies 
are needed. 

Future Studies 
The results presented in this dissertation lay the 
ground work for future studies with immense possibilities. 
With the help of a strain with a conditional fucosylation 
mutation, I was able to recognize the presence of a 
fucosylation pathway that otherwise may have gone 
undetected. Future research should focus on FP21 or the 
cytosolic fucosyltransf erase. 

Studies on FP21 

The first question to be addressed will be the 
compartmentalization of FP21 using an independent approach 
from that followed in my studies. An initial step would be 
to raise antibodies against FP21. A protocol to purify FP21 
is being improved in the laboratory, and should prove useful 
for this purpose. An antibody against FP21 will be useful 
for immunolocalization of the acceptor in fixed cells. 
Currently, FP21 is detected by SDS-PAGE as a fucose-labelled 
21 kD MW species. In the P100, the presence of other 
fucoconjugates of similar MW on SDS-PAGE could mask FP21, 
although, as discussed earlier, it appears that FP21 is not 
present in the P100 fraction. However, if FP21 was entirely 
released into the S100 fraction during cell fractionation 



150 
that would explain its absence in the P100. Alternatively, 
a higher MW precursor may exist in the P100. 
Immunolocalization of FP21 will help clarify this point. An 
antibody against FP21 will help in localizing FP21 in other 
compartments of the cell, if present, such as in the nucleus 
or nuclear membranes. 

Another aspect of interest is the other sugar residues 
present in the FP21 oligosaccharide. Due to its size (4.8 
glucose units) I suspect the carbohydrate moiety is truly an 
oligosaccharide, containing more than one sugar residue. 
There is evidence for a peptide-GlcNAc transferase in the 
cytosol of rat hepatocytes (Haltiwanger et al., 1990), so it 
is possible that the oligosaccharide is O-linked to the 
polypeptide backbone via a GlcNAc residue. The fact that 
the FP21 radioactive peak released by alkaline hydrolysis 
was not symmetric, suggested there is more than one type of 
oligosaccharide. The first step would be to separate and 
purify the oligosaccharides. For this purpose, a longer P-4 
column could be used. Alternatively, the oligosaccharides 
could be separated by other chromatographic methods 
(Townsend et al., 1989; Beniak et al., 1988). Once 
separated, the oligosaccharides can be examined by nuclear 
magnetic resonance spectrometry. 

Fucosylated FP21 was present in the cytosol of 
vegetative and developing cells, but at this moment the 
relative levels of expression at different developmental 



151 
times, nor if its preferentially expressed in any cell type 
during development, have been determined. Ideally, it would 
be useful to produce antibodies with specificity for the 
glycosylated protein, and specificity for the peptide moiety 
of FP21 (similar to other mAb produced in the laboratory; 
see West et al., 1986). Using these antibodies, 
fucosylation of FP21 during development could be followed by 
immunoprecipitation of FP21. 

Studies on the Cytosolic Fucosyltransferase 

Another aspect of my project was the evidence presented 
for a novel fucosyltransferase that appears to be cytosolic 
and seems to differ from the bulk sedimentable 
fucosyltransferase activity. The first question to be 
addressed will be the compartmentalization of the enzyme. 
The cytosolic enzyme could be purified by conventional 
methods (Beyer et al., 1980; Foster et al., 1991; Martin et 
al., 1987). Once purified, antibodies could be raised 
against the enzyme and used for immunolocalization of the 
fucosyltransferase. Currently, a purification protocol is 
being developed in the laboratory. If localization of the 
enzyme is done by immunofluorescence and the enzyme is a 
soluble cytosolic protein, it should be possible to observe 
a cytosolic distribution of the enzyme and an absence from 
intracellular vesicles. However, if the immunofluorescence 
pattern shows labelling of vesicles, the results will need 



152 
to be examined more carefully. It is possible that the 
enzyme fucosylates the cytosolic acceptor, FP21, while being 
membrane-associated, but facing the cytosol. There is a 
membrane-associated glycosyltransferase that utilizes 
cytosolic acceptors (Haltiwanger et al., 1990). If a 
portion of the fucosyltransferase pool was to partition to 
the outside of the vesicles, the activity on intact vesicles 
should fucosylate the type I analog. Fucosylation of this 
synthetic acceptor by intact P100 vesicles should be 
dependent on added Mg ++ . 

Another approach to study the cytosolic 
fucosyltransferase is to clone and seguence the enzyme, 
avoiding purification of the protein. To date, only two 
fucosyltransferases have been seguenced, one encodes an 
al, 3/1, 4fucosyl transferase and the other an 

al,3fucosyltransf erase (Kukowska-Latallo et al., 1990; Goelz 
et al., 1990). There is 57% identity between the two 
enzymes at the C-terminus, for a stretch of two-thirds the 
length of the protein (Goelz et al., 1990). Both enzymes 
appear to be type II transmembrane proteins, each composed 
of a short amino-terminal cytoplasmic domain with no 
discernible signal seguence, and a putative single 
transmembrane signal/anchor domain (Kukowska-Latallo et al., 
1990; Goelz et al., 1990). The seguenced 

fucosyltransferases possessed N-linked glycosylation sites, 
and one of them was shown to be a glycoprotein (Kukowska- 



153 
Latallo et al., 1990). Since the fucosyltransf erase 
reported in my studies appears to be cytosolic, it would be 
important to determine what is the relationship between 
microsomal and cytosolic fucosyltransf erases. All 
fucosyltransferases utilize the same sugar nucleotide donor, 
GDP-fucose, so it is likely that the GDP-fucose binding site 
would be similar for all enzymes. In addition, comparisons 
among the fucosyltransferases may reveal important 
information regarding intracellular targeting and possible 
evolutionary relationships. There is evidence that a 
retaining sequence allows glycosyltransferases to remain in 
the Golgi apparatus and endoplasmic reticulum (Paulson and 
Colley, 1989). The fact that the fucosyltransf erase 
reported in these studies localizes to the cytosol raises 
the possibility that the fucosyltransferase would lack the 
targeting and retaining sequences. 

In order to compare the cytosolic fucosyltransferase 
with the sequenced fucosyltransferases (Kukowska-Latallo et 
al., 1990; Goelz et al., 1990), it will be necessary to 
sequence the cytosolic fucosyltransferase. The 
aforementioned enzymes were cloned using a gene transfer 
system in which cloned cDNAs determined the expression of 
the enzyme in a recipient host that did not express such 
activity. It could be possible to do the same for the 
cytosolic fucosyltransferase, using the type I analog 
synthetic acceptor to screen for activity of transfected 



154 
clones. A suitable host to express the cytosolic 
fucosyltransf erase cDNA would be a mutant Dictyostelium 
strain, although there are no such mutants available at the 
present. On the other hand, yeast could be used, since it 
has been shown yeast cells do not carry out fucosylation 
(Kukuruzinska et al., 1987). One of the complications that 
may arise in trying to screen for clones expressing the 
cytosolic fucosyltransferase is the transfection of 
microsomal fucosyltransf erases. It remains to be determined 
whether the P100 fucosyltransferase activity is capable of 
fucosylating the type I analog. If the activity in the P100 
does not utilize the type I analog [galii( l,3)GlcNAcfi-8- 
methoxycarbonyloctyl ] as acceptor, then clones can be 
screened using the synthetic acceptor. However, if there 
are fucosyltransferases in the P100 that utilize the type I 
analog as acceptor, it will be necessary to differentiate 
the activity in vitro before the transfection experiments. 
P100 extracts will be assayed for the ability to fucosylate 
galfil,3GlcNAcfl-8-methoxycarbonyloctyl. If the fraction 
fucosylates the acceptor, the sensitivity to cations will be 
examined for possible differences with the cytosolic 
fucosyltransferase activity. If the fraction is active in 
the presence of EDTA, fucosyltransferase positive clones may 
be screened in the presence and absence of Mg ++ . Those that 
express activity only in the presence of Mg ++ may represent 
positive clones. In the event that the activity in the P100 



155 
is dependent on Mg ++ in a fashion similar to the cytosolic 
fucosyltransferase, other inhibitors should be tried, 
including tunicamycin and N-ethylmaleimide (Galland et al., 
1988; Campbell and Stanley; 1984). 



REFERENCES 



Abeijon, C. and C.B. Hirschberg. 1990. Topography of 

initiation of N-glycosylation reactions. J Biol. Chem. 
265:14691-14695. 

Aparicio, J., G.W. Erdos, and CM. West. 1990. Spore coat is 
altered in mod B glycosylation mutants of Dictyostelium 
discoideum. J. Cell. Biochem. 42:255-266. 

Arion, W.J., L.M. Ballas, A.J. Lange, and B.K. Wallin. 1976. 
Microsomal membrane permeability and the hepatic 
glucose-6-phosphatase system. Interactions of the 
system with D-mannose- 6 -phosphate and D-mannose. J. 
Biol. Chem. 251:4901-4907. 

Barondes, S.H. and P.L. Haywood. 1979. Comparison of 

developmental ly regulated lectins from three species of 
cellular slime mold. Biochim. Biophys. Acta 550:297- 
308. 

125 

Bartles, J.R. and W.A. Frazier. 1980. Preparation of I- 
discoidin I and the properties of its binding to 
Dictyostelium discoideum cells. J. Biol. Chem. 255: BO- 
Beniak, B., J. Orr, I. Brockhausen, G. Vella, and C. Phoebe. 
1988. Separation of neutral reducing oligosaccharides 
derived from glycoproteins by HPLC on a hydroxylated 
polymeric support. Anal. Biochem. 175:96-105. 

Bennett, G., C.P. Leblond, and A. Haddad. 1974. Migration of 
glycoprotein from the Golgi apparatus to the surface of 
various cell types as shown by radioautography after 
labeled fucose injection into rats. J. Cell Biol. 
60:259-284. 

Beyer, T.A. and R.L. Hill. 1980. Enzymatic properties of the 
J5-galactoside al-2 fucosyltransferase from porcine 
submaxillary gland. J. Biol. Chem. 255:5373-5379. 

Beyer, T.A., J.E. Sadler, and R.L. Hill. 1980. Purification 
to homogeneity of the H blood group B-galactoside al-2 
fucosyltransferase from porcine submaxillary gland. J. 
Biol. Chem. 255:5364-5372. 

156 



157 

Biermann, C.J. 1988. Hydrolysis and other cleavages of 

glycosidic linkages in polysaccharides. Adv. Carbohydr. 
Chem. Biochem. 46:251-271. 

Borts, R.H. and R.L. Dimond. 1981. The a-glucosidases of 
Dictyostelium discoideum. Develop. Biol. 87:176-184. 

Campbell, C. and P. Stanley. 1984. The Chinese hamster ovary 
glycosylation mutants LECH and LEC12 express two novel 
GDP-fucose:N-acetylglucosaminide 3-a-L- 
fucosyltransf erase enzymes. J. Biol. Chem. 259:11208- 
11214. 

Chen, P.S., Jr., T.Y. Toribara, and H. Warner. 1956. 
Microdetermination of phosphorus. Analyt. Chem. 
28:1756-1758. 

Chu, F.K. 1986. Requirements of cleavage of high mannose 
oligosaccharides in glycoproteins by peptide N- 
glycosidase F. J. Biol. Chem. 261:172-177. 

Darnell, J., H. Lodish, and D. Baltimore. 1986. Molecular 
Cell Biology. Scientific American Books, Inc., New 
York. 

Das, O.P. and E.J. Henderson. 1986. A novel technique for 
gentle lysis of eukaryotic cells: isolation of plasma 
membranes from Dictyostelium discoideum. Biochim. 
Biophys. Acta 736:43-56. 

Davis, L.I. and G. Blobel. 1987. Nuclear pore complex 

contains a family of glycoproteins that includes p62: 
glycosylation through a previously unidentified 
cellular pathway. Proc. Natl. Acad. Sci. USA 84:7552- 
7556. 

Dimond, R.L., R.A. Burns, and K.B. Jordan. 1981. Secretion 
of lysosomal enzymes in the cellular slime mold 
Dictyostelium discoideum. J. Biol. Chem. 256:6565-6572. 

Erdos, G.W. and CM. West. 1989. Formation and organization 
of the spore coat of Dictyostelium discoideum. Exp. 
Mycol. 13:169-182. 

Erdos, G.W. and D. Whitaker. 1983. Failure to detect 

immunocytochemically reactive endogenous lectin on the 
cell surface of Dictyostelium discoideum. J. Cell Biol. 
97:993-1000. 



158 

Finne, J., T. Krusius, R.K. Margolis, and R.U. Margolis. 
1979. Novel mannitol-containing oligosaccharides 
obtained by mild alkaline borohydride treatment of a 
chondroitin sulfated proteoglycan from brain. J. Biol. 
Chem. 254:10295-10300. 

Flowers, H.M. 1981. Chemistry and biochemistry of D- and L- 
fucose. Adv. Carbohydr. Chem. Biochem. 39:279-345. 

Foster, C.S., D.R.B. Gillies, and M.C. Glick. 1991. 

Purification and characterization of GDP-L-fuc-N- 
acetyl-fl-D-glucosaminide al-3fucosyltransferase from 
human neuroblastoma cells. Unusual substrate 
specificities of the tumor enzyme. J. Biol. Chem. 
266:3526-3531. 

Franke, J. and R. Kessin. 1977. A defined minimal medium for 
axenic strains of Dictyostelium discoideum. Proc. Natl. 
Acad. Sci. USA 74:2157-2161. 

Galland, S., A. Degiuli, J. Frot-Coutaz, and R. Got. 1988. 
Transfer of N-acetylglucosamine to endogenous 
glycoproteins in the nucleus and in non-nuclear 
membranes of rat hepatocytes : electrophoretic analysis 
of the endogenous acceptors. Biochem. Intl. 17:59-67. 

Goelz, S.E., C. Hession, D. Goff, B. Griffiths, R. Tizard, 
B. Newman, G. Chi-Rosso, and R. Lobb. 1990. ELFT: a 
gene that directs the expression of an ELAM-1 ligand. 
Cell. 63:1349-1356. 

Goldberg, A.H., L.C. Yeoman, and H. Busch. 1978. Chromat in- 
associated glycoproteins of normal rat liver and 
Novikoff hepatoma ascites cells. Cancer Res. 38:1052- 
1056. 

Gonzalez-Yanes, B., R.B. Mandell, M. Girard, S. Henry, 0. 
Aparicio, M. Gritzalli, R.D. Brown, Jr., G.W. Erdos, 
and CM. West. 1989. The spore coat of a fucosylation 
mutant in Dictyostelium discoideum. Develop. Biol. 
133:576-587. 

Goodloe-Holland, CM. and E.J. Luna. 1987. Purification and 
characterization of Dictyostelium discoideum plasma 
membranes. Meth. Cell Biol. 28:215-229. 

Gregg, J.H. and G.C Karp. 1978. Patterns of cell 

differentiation revealed by L-[ 3 H]fucose incorporation 
in Dictyostelium. Exptl. Cell Res. 112:31-46. 



159 

Hallgreen, P., A. Lundblad, and S. Svensson. 1975. A new 

type of carbohydrate-protein linkage in a glycopeptide 
from normal human urine. J. Biol. Chem. 250:5312-5314. 

Haltiwanger, R.S., G.D. Holt, and G.W. Hart. 1990. Enzymatic 
addition of O-GlcNAc to nuclear and cytoplasmic 
proteins. J. Biol. Chem. 265:2563-2568. 

Hardy, M.R., R.R. Townsend, and Y.C. Lee. 1988. 

Monosaccharide analysis of glycoconjugates by anion 
exchange chromatography with pulsed amperometric 
detection. Anal. Biochem. 170:54-62. 

Hart, G.W., R.S. Haltiwanger, G.D. Holt, and W.G. Kelly. 
1989a. Glycosylation in the nucleus and cytoplasm. 
Annu. Rev. Biochem. 58:841-874. 

Hart, G.W., R.S. Haltiwanger, G.D. Holt, and W.G. Kelly. 
1989b. Nucleoplasmic and cytoplasmic glycoproteins. 
Ciba Found. Symp. 145:102-118. 

Henderson, P.J.F. 1985. Statistical analysis of enzyme 

kinetic data. In: Technigues in the Life Sciences, vol. 
Bl/II, Protein and Enzyme Biochemistry, BS114:l-48, 
Elsevier Scientific Publishers, Ltd., Ireland. 

Hirschberg, C.B., M. Perez, M.D. Snider, W.L. Hanneman, J. 
Esko, and C.R.H. Raetz. 1982. Autoradiographic 
detection and characterization of a Chinese hamster 
ovary cell mutant deficient in fucoproteins. J. Cell. 
Physiol. 111:255-263. 

Hirschberg, C.B. and M.D. Snider. 1987. Topography of 

glycosylation in the rough endoplasmic reticulum and 
golgi apparatus. Ann. Rev. Biochem. 56:63-87. 

Ivatt, R.L., O.P. Das, E.J. Henderson, and P.W. Robbins. 
1984. Glycoprotein biosynthesis in Dictyostelium 
discoideum: developmental regulation of the protein- 
linked glycans. Cell 38:561-567. 

Jackson, S.P. and R. Tjian. 1988. O-Glycosylation of 

eukaryotic transcription factors: implications for 
mechanisms of transcriptional regulation. Cell 55:125- 
133. 

Kan, F.W.K. and P. Pinto da Silva. 1986. Preferential 

association of glycoproteins to the euchromatin regions 
of cross-fractured nuclei is revealed by fracture- 
label. J. Cell Biol. 102:576-586. 



160 

Kawasaki, T. and I. Yamashina. 1972. Isolation and 

characterization of glycopeptides from rat liver 
nuclear membrane. J. Biochem. 72:1517-1525. 

Knecht, D.A., D.L. Fuller, and W.F. Loomis. 1987. Surface 
glycoprotein, gp24, involved in early adhesion. 
Develop. Biol. 121:277-283. 

Kohnken, R.E. and E.A. Berger. 1987. Assay and 

characterization of carbohydrate binding by the lectin 
discoidin I immobilized on nitrocellulose. Biochemistry 
25:3949-3957. 

Kornfeld, R.H. and V. Ginsburg. 1966. Control of synthesis 

of guanosine 5 ' -diphosphate D-mannose and guanosine 5'- 
diphosphate L-fucose in bacteria. Biochim. Biophys. 
Acta 117:79-87. 

Kornfeld, R. and S. Kornfeld. 1985. Assembly of asparagine- 
linked oligosaccharides. Annu. Rev. Biochem. 54:631- 
664. 

Kornfeld, K., M.L. Reitman, and R. Kornfeld. 1981. The 
carbohydrate-binding specificity of pea and lentil 
lectins. Fucose is an important determinant. J. Biol. 
Chem. 256:6633-6640. 

Kukowska-Latallo, J.F., R.D. Larsen, R.P. Nair, and J.B. 

Lowe. 1990. A cloned human cDNA determines expression 
of a mouse stage-specific embryonic antigen and the 
Lewis blood group (al,3/al,4) fucosyltransf erase. Genes 
Dev. 4:1288-1303. 

Kukuruzinska, M.A., M.L.E. Bergh, and B.J. Jackson. 1987. 
Protein glycosylation in yeast. Ann. J?ev. Biochem. 
56:915-944. 

Kumazaki, T. and A. Yoshida. 1984. Biochemical evidence that 
secretor gene, Se, is a structural gene encoding a 
specific fucosyltransf erase. Proc. Natl. Acad. Sci. USA 
81:4193-4197. 

Lam, T.Y. and C.-H. Siu. 1981. Synthesis of stage-specific 
glycoproteins in Dictyostelium discoideum during 
development. Develop. Biol. 83:127-137. 

Levy-Wilson, B. 1983. Glycosylation, ADP-ribosylation, and 
methylation of Tetrahymena histones. Biochemistry 
22:484-489. 



161 

Lewin, A.S., V. Hines, and G.M. Small. 1990. Citrate 

synthase encoded by the CIT2 gene of Saccharomyces 
cerevisiae is peroxisomal. Mol. Cell Biol. 10:1399- 
1405. 

Liao, T.-H. and G.A. Barber. 1971. The synthesis of 

guanosine 5 ' -diphosphate L-fucose by enzymes of a 
higher plant. Biochim. Biophys. Acta 230:64-71. 

Lichtsteiner, S. and U. Schibler. 1989. A glycosylated 
liver-specific transcription factor stimulated 
transcription of the albumin gene. Cell 57:1179-1187. 

Lis, H. and N. Sharon. 1986. Lectins as molecules and as 
tools. Annu. Rev. Biochem. 55:35-67. 

Loomis, W.F. 1971. Sensitivity of Dictyostelium discoideum 
to nucleic acid analogues. Exptl. Cell Res. 64:484-486 

Loomis, W.F. 1987. Genetic tools for Dictyostelium 
discoideum. Methods Cell Biol. 28:31-65. 

Magner, J. A., W. Novak, and E. Papagiannes. 1986. 

Subcellular localization of fucose incorporation into 
mouse thyrotropin and free a-subunits: studies 
employing subcellular fractionation and inhibitors of 
the intracellular translocation of proteins. 
Endocrinology 119:1315-1328. 

Margolis, R.U. , K. Lalley. W.-L. Kiang, C. Crockett, and 
R.K. Margolis. 1976. Isolation and properties of a 
soluble chondroitin sulfate proteoglycan from brain. 
Biochem. Biophys. Res. Comm. 73:1018-1024. 

Martin, A., M.-C. Biol, M. Richard, and P. Louisot. 1987. 
Purification and separation of two soluble 
fucosyltransferase activities of small intestinal 
mucosa. Comp. Biochem. Physiol. 87B: 725-731. 

McMahon, D. , M. Miller, and S. Long. 1977. The involvement 
of the plasma membrane in the development of 
Dictyostelium discoideum. Biochim. Biophys. Acta 
465:224-241. 

Nicolson, G., M. Lacorbiere, and P. Delmonte. 1972. Outer 
membrane terminal saccharides of bovine liver nuclei 
and mitochondria. Exptl. Cell Res. 71:468-473. 

Nunez, H.A. and R. Barker. 1976. The metal ion catalyzed 
decomposition of nucleoside diphosphate sugars. 
Biochemistry 15:3843-3847. 



162 

Palcic, M.M., L.D. Heerze, M. Pierce, and 0. Hindsgaul. 

1988. The use of hydrophobic synthetic glycosides as 
acceptors in glycosyl transferase assays. Glycocon jugate 
J. 5:49-63. 

Paulson, J.C. and K.J. Colley. 1989. Glycosyl transf erases . 
Structure, localization, and control of cell type- 
specific glycosylation. J. Biol. Chem. 264:17615-17618. 

Paulson, J.C, J. -P. Prieels, L.R. Glasgow, and R.L. Hill. 
1978. Sialyl- and fucosyltransf erases in the 
biosynthesis of asparaginyl-linked oligosaccharides in 
glycoproteins. Mutually exclusive glycosylation by R- 
galactoside a2-6 sialyltransferase and N- 
acetylglucosaminide al-3 fucosyltransf erase. J. Biol. 
Chem. 253:5617-5624. 

Perez, M. and C.B. Hirschberg. 1986. Transport of sugar 
nucleotides and adenosine 3 ' -phosphate 5 1 - 
phosphosulfate into vesicles derived from the Golgi 
apparatus. Biochim. Biophys. Acta 864:213-222. 

Poole, S., R.A. Firtel, and E. Lamar. 1981. Sequence and 
expression of the discoidin I gene family in 
Dictyostelium discoideum. J. Mol. Biol. 153:273-289. 

Potvin, B., R. Kumar, D.R. Howard, and P. Stanley. 1990. 

Transf ection of a human a-( 1,3) fucosyltransf erase gene 
into Chinese hamster ovary cells. Complication arise 
from activion of endogenous a-( 1,3) fucosyltransf erases. 
J. Biol. Chem. 265:1615-1622. 

Rajan, V.P., R.D. Larsen, S. Ajmera, L.K. Ernst, and J.B. 
Lowe. 1989. A cloned human DNA restriction fragment 
determines expression of a GDP-L-fucose:fi-D-galactoside 
2-a-L-fucosyltransf erase in transf ected cells. J. Biol. 
Chem. 264:11158-11167. 

Reeves, R. and D. Chang. 1983. Investigations of the 
possible functions for glycosylation in the high 
mobility group proteins. Evidence for a role in nuclear 
matrix association. J. Biol. Chem. 258:679-687. 

Reeves, R., D. Chang, and S.-C. Chung. 1981. Carbohydrate 
modifications of the high mobility group proteins. 
Proc. Natl. Acad. Sci. USA 78:6704-6708. 

Reitman, M.L., I.S. Trowbridge, and S. Kornfeld. 1980. Mouse 
lymphoma cell lines resistant to pea lectin are 
defective in fucose metabolism. J. Biol. Chem. 
255:9900-9906. 



163 

Richard, M., A. Martin, and P. Louisot. 1975. Evidence for 
glycosyl-transf erases in rat liver nuclei. Biochem. 
Biophys. Res. Comm. 64:108-114. 

Ripka, J., A. Adamany, and P. Stanley. 1986. Two Chinese 
hamster ovary glycosylation mutants affected in the 
conversion of GDP-mannose to GDP-fucose. Arch. Biochem. 
Biophys. 249:533-545. 

Ripka, J. and P. Stanley. 1986. Lectin-resistant CHO cells: 
selection of four new pea lectin-resistant phenotypes. 
Somatic Cell Mol . Genet. 12:51-62. 

Satir, B.H., C. Srisomsap, M. Reichman, and R.B. Marchase. 
1990. Parafusin, an exocytic-sensitive phosphoprotein, 
is the primary acceptor for the 

glucosylphosphotransf erase in Paramecium tetraurelia 
and rat liver. J. Cell Biol. 111:901-907. 

Scrimgeour, K.G. 1977. Chemistry and Control of Enzyme 
Reactions. Academic Press, London and New York. 

Seve, A. -P., J. Hubert, D. Bouvier, C. Bourgeois, P. Midoux, 
A.-C. Roche, and M. Monsigny. 1986. Analysis of sugar- 
binding sites in mammalian cell nuclei by quantitative 
flow microfluorometry. Proc. Natl. Acad. USA 83:5997- 
6001. 

Snider, M.D., L.A. Sultzman, and P.W. Robbins. 1980. 
Transmembrane location of oligosaccharide-lipid 
synthesis in microsomal vesicles. Cell 21:385-392. 

Sommers, L.W. and C.B. Hirschberg. 1982. Transport of sugar 
nucleotides into rat liver Golgi. A new Golgi marker 
activity. J. Biol. Chem. 257:10811-10817. 

Spielman, J., S.R. Hull, Z.Q. Sheng, R. Kanterman, A. 

Bright, K.L. Carraway. 1988. Biosynthesis of a tumor 
cell surface sialomucin. Maturation and effects of 
monensin. J. Biol. Chem. 263:9621-9629. 

Stanley, P. 1984. Glycosylation mutants of animal cells. 
Ann. Rev. Genet. 18:525-552. 

Stein, G.S., R.M. Roberts, J.L. Davis, W.J. Head, J.L. 

Stein, C.L. Thrall, J. van Veen, and D.W. Welch. 1975. 
Are glycoproteins and glycosaminoglycans components of 
the eukaryotic genome? Nature 258:639-641. 

Stone, D.B., P.M.G. Curmi, and R.A. Mendelson. 1987. 

Preparation of deuterated actin from Dictyostelium 
discoideum. Meth. Cell Biol. 28:215-229. 



164 

Stroup, G.B., K.R. Anumula, T.F. Kline, and M.M. Caltabiano. 
1990. Identification and characterization of two 
distinct a-( 1-3) -L-fucosyltransferase activities in 
human colon carcinoma. Cancer Res. 50:6787-6792. 

Tarentino, A.L., CM. Gomez, and T.H. Plummer, Jr. 1985. 
Deglycosylation of asparagine-linked glycans by 
peptide :N-glycosidase F. Biochemistry 24:4665-4671. 

Townsend, R.R., M.R. Hardy, D.A. dimming, J. P. Carver, and 
B. Bendiak. 1989. Separation of branched sialylated 
oligosaccharides using high-pH anion-exchange 
chromatography with pulsed amperometric detection. 
Anal. Biochem. 182:1-8. 

Tschursin, E., G.R. Riley, and E.J. Henderson. 1989. 

Differential regulation of glycoprotein sulfation and 
fucosylation during growth of Dictyostelium discoideum. 
Differentiation 40:1-9. 

Virtanen, I. and J. Wartiovaara. 1976. Lectin receptor sites 
on rat liver cell nuclear membranes. J. Cell Sci. 
22:335-344. 

Weinstein, J., E.U. Lee, K. McEntee, P.-H. Lai, and J.C. 

Paulson. 1987. Primary structure of fi-galactoside ct2,6- 
sialyltransf erase. Conversion of membrane-bound enzyme 
to soluble forms by cleavage of the NH 2 -terminal signal 
anchor. J. Biol. Chem. 262:17735-17743. 

West, CM. and S.A. Brownstein. 1988. EDTA treatment alters 
protein glycosylation in the cellular slime mold 
Dictyostelium discoideum. Exptl. Cell Res. 175:26-36. 

West, CM. and G.W. Erdos. 1988. The expression of 

glycoproteins in the extracellular matrix of the 
cellular slime mold Dictyostelium discoideum. Cell 
Differ. 23:1-6. 

West, CM. and G.W. Erdos. 1990. Formation of the 

Dictyostelium spore coat. Dev. Genet. 11:492-506. 

West, CM., G.W. Erdos, and R. Davis. 1986. Glycoantigen 

expression is regulated both temporally and spatially 
during development in the cellular slime molds 
Dictyostelium discoideum and L. mucoroides. Mol. Cell. 
Biochem. 72:121-140. 



165 

West, CM. and W.F. Loomis. 1985. Absence of a carbohydrate 
modification does not affect the level or subcellular 
localization of three membrane glycoproteins in modB 
mutants of Dictyostelium discoideum. J. Biol. Chem. 
260:13803-13809. 

Yamashita, K., T. Mizuochi, and A. Kobata. 1982. Analysis of 
oligosaccharides by gel filtration. Meth. Enzymol. 
83:105-126. 

Yurchenko, P.D. and P.H. Atkinson. 1975. Fucosyl- 

glycoprotein and precursor pools in HeLa cells. 
Biochemistry 14:3107-3114. 

Yurchenko, P.D., C. Ceccarini, and P.H. Atkinson. 1978. 
Labeling complex carbohydrates of animal cells with 
monosaccharides. Meth. Enzymol. 50:175-204. 

Zatz, M. and S.H. Barondes. 1971. Particulate and 

solubilized fucosyltransferases from mouse brain. J. 
Neurochem. 18:1625-1637. 

Zinn, A.B., J.S. Marshall, and D.M. Carlson. 1978. 

Preparation of glycopeptides and oligosaccharides from 
thyroxine-binding globulin. J. Biol. Chem. 253:6761- 
6767. 



BIOGRAPHICAL SKETCH 
Beatriz Yadira Gonzalez-Yanes was born June 9, 1964, in 
Fajardo, Puerto Rico. She graduated from Nuestra Sefiora del 
Pilar High School in Rio Piedras, Puerto Rico, in 1981. 
Following high school, she attended the University of Puerto 
Rico in Rio Piedras, and earned a Bachelor of Science 
degree, Magna Cum Laude, in biology in 1985. In August 1985 
she entered graduate school at the University of Florida, 
and joined the Department of Anatomy and Cell Biology in 
February 1987. She completed the reguirements for the 
degree of Doctor of Philosophy in December 1991. She has 
accepted a postdoctoral research position in the Animal 
Science Department at the University of Florida in 
Gainesville, Florida. 



166 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 



(7\x- 



ML)aJT 



Christopher M. West, Chair 
Associate Professor of Anatomy and 
Cell Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

Carl M. Feldherr 

Professor of Anatomy and Cell 

Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
a dissertation for the degree of Doctor of Philosophy. 



(jJixL^o 




as 



William A. Dunn, Jr./ 

Assistant Professor of Anatomy and 

Cell Biology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 



PffcA f(7 ^Cv, 



Robert J. Cohen 

Associate Professor of Biochemistry 

and Molecular Biology 



This dissertation was submitted to the Graduate Faculty 
of the College of Medicine and to the Graduate School and 
was accepted as partial fulfillment of the requirements for 
the degree of Doctor of Philosophy. 



December 1991 




*JH 




Dean, College of Medicine 
idua 



• ' \ <rt ^f.j^-cL,^^ (J^~<^*^^t<-ci-^.yf 



Dean, Graduate School